Methods and compositions for the treatment of proliferative disorders

ABSTRACT

The invention features methods of treating a proliferative disorder characterized by elevated Pin1 marker levels and/or reduced Pin1 Ser71 phosphorylation in a subject by administering a retinoic acid compound. Additionally, the invention features methods of treating proliferative disorders (e.g., proliferative disorders characterized by elevated Pin1 marker levels) by administering a retinoic acid compound in combination with another anti-proliferative compound. Finally, the invention also features methods including high-throughput screens for discovering and validating Pin1 inhibitors.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbers NIHGM058556, NIH AG017870, and NIH CAl22434. The government has certainrights in the invention.

FIELD OF THE INVENTION

In general, this invention relates to the treatment of proliferativedisorders (e.g., proliferative disorders characterized by elevated Pin1marker levels) with retinoic acid compounds.

BACKGROUND OF THE INVENTION

The increased number of cancer cases reported in the United States, and,indeed, around the world, is a major concern. Currently there are only ahandful of detection and treatment methods available for some specifictypes of cancer, and these provide no absolute guarantee of success. Inorder to be most effective, these treatments require not only an earlydetection of the malignancy, but a reliable assessment of the severityof the malignancy.

It is apparent that the complex process of tumor development and growthmust involve multiple gene products. It is therefore important to definethe role of specific genes involved in tumor development and growth andidentify those genes and gene products that can serve as targets for thediagnosis, prevention, and treatment of cancers.

In the realm of cancer therapy, it often happens that a therapeuticagent that is initially effective for a given patient becomes, overtime, ineffective or less effective for that patient. The very sametherapeutic agent may continue to be effective over a long period oftime for a different patient. Further, a therapeutic agent that iseffective, at least initially, for some patients can be completelyineffective or even harmful for other patients. Accordingly, it would beuseful to identify genes and/or gene products that represent prognosticgenes with respect to a given therapeutic agent or class of therapeuticagents. It then may be possible to determine which patients will benefitfrom particular therapeutic regimen and, importantly, determine when, ifever, the therapeutic regime begins to lose its effectiveness for agiven patient. The ability to make such predictions would make itpossible to discontinue a therapeutic regime that has lost itseffectiveness well before its loss of effectiveness becomes apparent byconventional measures.

Recent advances in the understanding of molecular mechanisms ofoncogenesis have led to exciting new drugs that target specificmolecular pathways. These drugs have transformed cancer treatments,especially for those caused by some specific oncogenic events, such asHerceptin for breast cancer, caused by HER2/Neu, and Gleevec for chronicmyelogenous leukemia caused by Bcr-Abl. However, it has beenincreasingly evident that, in many individual tumors, there are a largenumber of mutated genes that disrupt multiple interactive and/orredundant pathways. Thus, intervening in a single pathway may not beeffective. Furthermore, cancer resistance to molecularly targeted drugscan develop through secondary target mutation or compensatory activationof alternative pathways, so-called “oncogenic switching.” Thus, a majorchallenge remains how to simultaneously inhibit multiple oncogenicpathways either using a combination of multiple drugs, with each actingon a specific pathway, or using a single drug that concurrently blocksmultiple pathways. The results disclosed herein suggest that Pin1inhibitors might have a major impact on treating cancers, especiallyaggressive and/or drug-resistant cancers.

We and others have shown that Pin1 is prevalently overexpressed in humancancers and that high Pin1 marker levels correlate with poor clinicaloutcome in many cancers. In contrast, the Pin1 polymorphism that reducesPin1 expression is associated with reduced cancer risk in humans.Significantly, Pin1 activates at least 19 oncogenes/growth enhancers,including β-catenin, cyclin D1, NF-κB, c-Jun, c-fos, AKT, A1B1,HER2/Neu, MCl-1, Notch, Raf-1, Stat3, c-Myb, Hbx, Tax, and v-rel, andalso inactivates at least 12 tumor suppressors/growth inhibitors,including PML, SMRT, FOXOs, RARα, and Smad (FIGS. 1A and 1B). WhereasPin1 overexpression causes cell transformation and tumorigenesis, Pin1knockdown inhibits cancer cell growth in cell cultures and mice.Pin1-null mice are highly resistant to tumorigenesis induced either byoncogenes such as activated Ras or HER2/Neu, or tumor suppressors suchas p53. Thus, Pin1 inhibitors might have the desired property tosuppress numerous oncogenic pathways simultaneously for treatingcancers, especially those aggressive and/or drug-resistant cancers.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of treating aproliferative disease in a subject by administering a retinoic acidcompound (e.g., a deuterated compound) to the subject in an amountsufficient to treat the subject, wherein the subject is determined tohave elevated levels of a Pin1 marker (e.g., Ser71 phosphorylation)prior to the administration.

In another aspect, the invention features a method of treating aproliferative disease in a subject by determining Pin1 marker levels(e.g., reduced Ser71 phosphorylation) in a sample (e.g., tumor samples,blood, urine, biopsies, lymph, saliva, phlegm, and pus) from the subjectand administering a retinoic acid compound to the subject if sample isdetermined to have elevated Pin1 marker levels.

In any of the foregoing aspects, the method can also include theadministration of a second anti-proliferative compound (e.g., at a lowdosage) or anti-cancer compound (e.g., an anti-angiogenic compound). Thesecond compound can be administered separately, or in a singleformulation with, the retinoic acid compound. The secondanti-proliferative compound can be, e.g., MK-2206, ON 013105, RTA 402,BI 2536, Sorafenib, ISIS-STAT3Rx, a microtubule inhibitor, atopoisomerase inhibitor, a platin, an alkylating agent, ananti-metabolite, paclitaxel, gemcitabine, doxorubicin, vinblastine,etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide,amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine,chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine,cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin,daunorubicin, docetaxel, estramustine phosphate, floxuridine,fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide,imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen,6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone,pentostatin, procarbazine, rituximab, streptozocin, tamoxifen,temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab,vincristine, vindesine, and/or vinorelbine. Additionally, oralternatively, any of the foregoing methods can include determining Pin1marker levels in the sample after the administration of a retinoic acidcompound.

In any of the foregoing aspects, the retinoic acid compound may selectedfrom 13-cis-retinoic acid, all-trans-retinoic acid, retinol, retinylacetate, retinal, AC-55649, acitretin or any of the compounds listed inFIG. 2A, FIG. 2B, FIG. 10A, FIG. 10D, and Table 1.

The elevated Pin1 marker level of any of the foregoing methods can bedue to, e.g., an inherited trait or a somatic mutation.

The proliferative disorder of any of the foregoing methods can be, e.g.,leukemias, polycythemia vera, lymphomas, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors. Specifically,the proliferative disorder can be e.g., acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemia, acute myeloblasticleukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia,acute monocytic leukemia, acute erythroleukemia, chronic leukemia,chronic myelocytic leukemia, chronic lymphocytic leukemi), Hodgkin'sdisease, non-Hodgkin's disease, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma.

The invention also features a method for identifying a Pin1 ligandincluding: (i) incubating Pin1 protein with a fluorescently labeledprobe (e.g., HF488-FP or FITC-FP labeled peptide probes), forming aPin1-probe complex; (ii) adding a test compound to the incubation; and(iii) determining whether any substantial portion of the probe isdisplaced from the Pin1-probe complex by the test compound, suchdisplacement indicating that the test compound is a Pin1 ligand. Theinvention also features a method for identifying a Pin1 modulatingcompound including: (i) incubating human-derived cancer cells (e.g.,breast cancer cells) that have Neu/Erb2 gene amplification; (ii)applying a test compound to the cell; and (iii) determining whetherNeu/Erb2 overexpression is reduced, wherein reduction of Neu/Erb2overexpression indicates that the test compound is a Pin1 modulatingcompound.

By the term “proliferative disorder” is meant a disorder characterizedby inappropriate accumulation of a cell population in a tissue (e.g., byabnormal cell growth). This inappropriate accumulation may be the resultof a genetic or epigenetic variation that occurs in one or more cells ofthe cell population. This genetic or epigenetic variation causes thecells of the cell population to grow faster, die slower, ordifferentiate slower than the surrounding, normal tissue. The cellpopulation includes cells of hematopoietic, epithelial, endothelial, orsolid tissue origin.

As used herein, the term “abnormal cell growth” is intended to includecell growth which is undesirable or inappropriate. Abnormal cell growthalso includes proliferation which is undesirable or inappropriate (e.g.,unregulated cell proliferation or undesirably rapid cell proliferation).Abnormal cell growth can be benign and result in benign masses of tissueor cells, or benign tumors. Many art-recognized conditions areassociated with such benign masses or benign tumors including diabeticretinopathy, retrolental fibrioplasia, neovascular glaucoma, psoriasis,angiofibromas, rheumatoid arthrtis, hemangiomas, and Karposi's sarcoma.Abnormal cell growth can also be malignant and result in malignancies,malignant masses of tissue or cells, or malignant tumors. Manyart-recognized conditions and disorders are associated withmalignancies, malignant masses, and malignant tumors including cancerand carcinoma.

As used herein, the term “tumor” is intended to encompass both in vitroand in vivo tumors that form in any organ of the body. Tumors may beassociated with benign abnormal cell growth (e.g., benign tumors) ormalignant cell growth (e.g., malignant tumors). The tumors which aredescribed herein are preferably sensitive to the Pin1 inhibitors of thepresent invention. Examples of the types of tumors intended to beencompassed by the present invention include those tumors associatedwith breast cancer, skin cancer, bone cancer, prostate cancer, livercancer, lung cancer, brain cancer, cancer of the larynx, gallbladder,pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head andneck, colon, stomach, bronchi, kidneys.

As used herein, the term “Pin1 marker” refers to a marker which iscapable of being indicative of Pin1 activity levels in a sample of theinvention. Pin1 markers include nucleic acid molecules (e.g., mRNA, DNA)which corresponds to some or all of a Pin1 gene, peptide sequences(e.g., amino acid sequences) which correspond to some or all of a Pin1protein, nucleic acid sequences which are homologous to Pin1 genesequences, peptide sequences which are homologous to Pin1 peptidesequences, antibodies to Pin1 protein, substrates of Pin1 protein,binding partners of Pin1 protein, and activity of Pin1.

By “elevated levels of a Pin1 marker” is meant a level of Pin1 markerthat is altered thereby indicating elevated Pin1 activity. “Elevatedlevels of a Pin1 marker” include levels at least 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%,1000%, or greater than, or 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100% less than the marker levels measuredin a normal, disease fee subject or tissue.

By the term “retinoic acid compound” is meant a compound that is either(a) the diterpene retinoic acid, or a derivative thereof, or (b) acompound having the structure R¹—Ar¹-L¹Ar²-L²-C(═O)R³ (Formula (I)).Exemplary retinoic acid compounds described herein (includingderivatives thereof) include the compounds identified in FIGS. 2A, 2B,10A, 10D, and Table 1. The term “diterpene retinoic acid” encompassesany stereoisomer of retinoic acid (e.g., the retinoic acid may be in theall-trans configuration (ATRA) or one or more of the double bonds may bein the cis configuration (e.g., 13-cis retinoic acid (13cRA)).Derivatives of the diterpene retinoic acid include reduced forms such asretinal, retinol, and retinyl acetate. In Formula (I), each of Ar¹ andAr² is, independently, optionally substituted aryl or an optionallysubstituted heteroaryl; R¹ is H, an optionally substituted alkyl group,an optionally substituted alkenyl group, or an optionally substitutedalkynyl group; each of L¹ and L² is selected, independently from acovalent bond, an optionally substituted C₁₋₁₀ alkylene, an optionallysubstituted C₂₋₁₀ alkenylene (e.g., —CH═CH—, —COCH═CH—, —CH═CHCO—, adienyl group, or a trienyl group), optionally substituted C₂₋₁₀alkynylene (e.g., —C≡C—), or —(CHR⁴)_(n)CONR⁵—, —NR⁵CO—, where n is 0 or1, R⁴ is H or OH, and R⁵ is H or optionally substituted alkyl; and R³ isH, OR⁴ or N(R⁴)², where each R⁴ is selected, independently, from H,optionally substituted alkyl, or optionally substituted heteroalkyl.

Any of the chemical groups, functional groups, or substituents describedherein may be deuterated if the chemical group, functional group, orsubstituent has —H. As used herein, when a particular position in acompound of this invention is designated as being “deuterated” or“having deuterium,” it is understood that the position containsdeuterium or includes deuterium (the element deuterium is represented bythe letter “D” in chemical structures and formulas and indicated with alower case “d” in chemical names, according to the Boughton system).When any of the position is deuterated, it is understood that theabundance of deuterium at that position is substantially greater thanthe natural abundance of deuterium, which is 0.015%. In certainembodiments, a composition of the invention has a minimum isotopicenrichment factor of at least 5 (0.075% deuterium incorporation), e.g.,at least 10 (0.15% deuterium incorporation). In other embodiments, acomposition has an isotopic enrichment factor of at least 50 (0.75%deuterium incorporation), at least 500 (7.5% deuterium incorporation),at least 2000 (30% deuterium incorporation), at least 3000 (45%deuterium incorporation), at least 4000 (60% deuterium incorporation),at least 4500 (67.5% deuterium incorporation), at least 5000 (75%deuterium incorporation), at least 5500 (82.5% deuterium incorporation),at least 6000 (90% deuterium incorporation), or at least 6600 (99%deuterium incorporation).

As used herein, the term “isotopic enrichment factor” refers to theratio of the isotopic abundance of a composition to the naturalabundance of the specified isotope. For example, deuterium has a naturalabundance of 0.015%. A compound with, for example, 45% deuteriumincorporation at a specified position, has an isotopic enrichment factorof 3000 at that site relative to the natural abundance of deuterium.

TABLE 1 List of compounds structurally similar to retinoic acid CIDIUPAC Other names 444795(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1- Retinoicacid; yl)nona-2,4,6,8-tetraenoic acid tretinoin; Vitamin A acid 25145416(2Z,4E,6Z,8Z)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 23275881(2Z,4Z,6E,8Z)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 12358678(2E,4E,6E,8Z)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1- CHEMBL44478;yl)nona-2,4,6,8-tetraenoic acid CHEBI:168407; AC- 540 10881132(2Z,4Z,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10638113(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 9861147(2E,4Z,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 9796370(2E,4Z,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1- 1tyr;(11Z)-retinoic yl)nona-2,4,6,8-tetraenoic acid acid; 11-cis-Retinoicacid 6603983 (2E,4Z,6E,8Z)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-Tocris-0695; Lopac- yl)nona-2,4,6,8-tetraenoic acid R-2625; Lopac-R-3255 6419708 (2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-9,13-di-cis-RA; yl)nona-2,4,6,8-tetraenoic acid 9,13-Di-cis-retinoicacid; 9-cis,13-cis- Retinoic acid 5282379(2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1- Isotretinoin;13-cis- yl)nona-2,4,6,8-tetraenoic acid Retinoic acid; Accutan 449171(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1- Alitretinoin;Panretin; yl)nona-2,4,6,8-tetraenoic acid 9-CIS-RETINOIC ACID 55383,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-Spectrum_001676; tetraenoic acid SpecPlus_000696; AC1L1KKH 543055662,4-dideuterio-7-methyl-3-(trideuteriomethyl)-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 543055659-[3,3-dideuterio-6,6-dimethyl-2-(trideuteriomethyl)cyclohexen-1-yl]-3,7-dimethylnona-2,4,6,8-tetraenoic acid 10566385(2E,4E,6Z,8E)-7-methyl-3-(trideuteriomethyl)-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10518761(2E,4E,6Z,8E)-7-methyl-9-(2,6,6-trimethylcyclohexen-1-yl)-3-(tritritiomethyl)nona-2,4,6,8-tetraenoic acid 10470200(2E,4Z,6Z,8E)-4,5-dideuterio-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10425032(2E,4E,6Z,8E)-4,5-dideuterio-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10357701(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-4,5-ditritiocyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10267048(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)-4,5-ditritionona-2,4,6,8-tetraenoic acid 10086398(2Z,4Z,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)-4,5-ditritionona-2,4,6,8-tetraenoic acid 10086397(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-3,4-ditritiocyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10063649(2E,4E,6Z,8E)-9-[2,6-dimethyl-6-(trideuteriomethyl)cyclohexen-1-yl]-3,7-dimethylnona-2,4,6,8-tetraenoic acid 10040620(2E,4E,6Z,8E)-9-(4,5-dideuterio-2,6,6-trimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid 10017935(2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)-4,5-ditritionona-2,4,6,8-tetraenoic acid 10017822(2E,4E,6Z,8E)-9-(3,4-dideuterio-2,6,6-trimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid 9995220(2E,4Z,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)-4,5-ditritionona-2,4,6,8-tetraenoic acid 9972327(2Z,4Z,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)-4,5-ditritionona-2,4,6,8-tetraenoic acid 9972326(2E,4Z,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)-4,5-ditritionona-2,4,6,8-tetraenoic acid 9839397(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)-5-tritionona-2,4,6,8-tetraenoic acid 6913160(2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)-5-Retinoic-11-t acid; tritionona-2,4,6,8-tetraenoic acid AC1OC7MJ; all-trans-(11-3H)- Retinoic acid 6913136(2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)- AC1OC7KP;4,5-ditritionona-2,4,6,8-tetraenoic acid Retinoic-11,12-t2 acid;11,12-3H- Retinoic acid 6913131(2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)- AC1OC7KA;5,6-ditritionona-2,4,6,8-tetraenoic acid Retinoic-10,11-t2 acid;all-trans-(10,11- 3H2)-Retinoic acid 6439661(2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 1342623,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-SHGAZHPCJJPHSC- tetraenoic acid SPLUINJESA-N; FDEFF7D13961B766CC9FE8A740623243 56684147(2E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,6,8-trienoic acid 542198083,6,7-trimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 539369743,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,6,8- trienoic acid53740187 3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6-trienoic acid 44725022(Z)-3-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]hept-2-enoicAC1Q2V68; (2Z)-3- acid [(E)-2-(2,6,6- trimethylcyclohex-1-en-1-yl)ethenyl]hept- 2-enoic acid 21590819(2Z,4E,8E)-3-methyl-7-methylidene-9-(2,6,6- CHEMBL182393trimethylcyclohexen-1-yl)nona-2,4,8-trienoic acid 11738545(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)deca-2,4,6,8-tetraenoic acid 10518336(2E,4E,8E)-3-methyl-7-methylidene-9-(2,6,6- CHEMBL426963trimethylcyclohexen-1-yl)nona-2,4,8-trienoic acid 10380944(2E,4E,6Z,8E)-3-ethyl-7-methyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10335106(2E,4E,6E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-CHEMBL487208 2,4,6-trienoic acid 10286439(2E,4E,6Z,8E)-7-ethyl-3-methyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10149682(2E,4E,6Z,8E)-3,6,7-trimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10041353(2E,4E,6E,8E)-3-ethyl-7-methyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 6439749(2E,4E,6E,8E)-9-(2-ethyl-6,6-dimethylcyclohexen-1-yl)-3,7- SRI 2712-24;2,4,6,8- dimethylnona-2,4,6,8-tetraenoic acid Nonatetracenoic acid,5496917 (2E,4Z,6E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-AC1NUZ8L 2,4,6-trienoic acid 5326825(2Z,4Z,6E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona- AC1NS1592,4,6-trienoic acid 41365243-[2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]hept-2-enoic acid AC1N4YDA135317 9-(2-ethyl-6,6-dimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid 5452537013-(2,6,6-trimethylcyclohexen-1-yl)trideca-2,4,6,8,10,12- hexaenoic acid54472611 4,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 54398880 3-methyl-5-[2-[2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclopenten-1-yl]penta-2,4-dienoic acid 5404475011-(2,6,6-trimethylcyclohexen-1-yl)undeca-2,4,6,8,10-pentaenoic acid538768523,7-dimethyl-9-(2,4,6,6-tetramethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 537905699-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 537431045,9-dimethyl-11-(2,6,6-trimethylcyclohexen-1-yl)undeca-2,4,6,8,10-pentaenoic acid 44579060(2E,4E,6Z,8E)-9-(2-butyl-6,6-dimethylcyclohexen-1-yl)-3,7- CHEMBL518436dimethylnona-2,4,6,8-tetraenoic acid 44393163(2Z,4E,8E)-7-methylidene-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,8-trienoic acid 25141345(2E,4E,6E,8E)-9-(2-butyl-6,6-dimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid 19609253(2E,4E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclopenten-1-yl]penta-2,4-dienoic acid 14731990(2E,4E,6E,8E)-7-methyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 11141121(2E,4E,6E,8E)-4,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10712359(2E,4E,6Z)-3-methyl-7-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]undeca-2,4,6-trienoic acid 10474100(2E,4E,6E,8E,10E,12E)-3,7,11-trimethyl-13-(2,6,6-trimethylcyclohexen-1-yl)trideca-2,4,6,8,10,12-hexanoic acid 10426543(E,4E)-3-methyl-4-[3-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclohex-2-en-1-ylidene]but-2-enoic acid 10358907(Z,4E)-3-methyl-4-[(4E)-3-methyl-4-[(2,6,6-trimethylcyclohexen-1-yl)methylidene]cyclohexa-2,5-dien-1-yl)methylidene) 10314319(2E,4E,6E,8E,10E)-5,9-dimethyl-11-(2,6,6-trimethylcyclohexen-CHEMBL225948 1-yl)undeca-2.4,6,8,10-pentaenoic acid 10286753(2E,4E,6Z,8E)-7-tert-butyl-3-methyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10266931(2E,4E,6Z)-3-methyl-7-[(E)-2-(2,6,6-trimethylcyclohexen-1- CHEMBL507779yl)ethenyl]deca-2,4,6-trienoic acid 10125803(2E,4E,6Z)-3-methyl-7-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]deca-2,4,6-trienoic acid 10087786(Z,4E)-3-methyl-4-[3-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclohex-2-en-1-ylidene]but-2-enoic acid 10015486(2E,4E,6E)-5-methyl-7-(2,6,6-trimethylcyclohexen-1-yl)hepta-2,4,6-trienoic acid 9929074(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 9860303(2E,4E,6E,8E)-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8- tetraenoicacid 5355027(2E,4E)-3-methyl-5-(2,6,6-trimethylcyclohexen-1-yl)penta-2,4- C15 acid;dienoic acid AC1NS6O9; NSC23978 1670953-methyl-5-(2,6,6-trimethylcyclohexen-1-yl)penta-2,4-dienoic acidAC1L4ZB4 566068323,7-dimethyl-9-(9,9,11-trimethylspiro[2.5]oct-10-en-10-yl)nona-2,4,6,8-tetraenoic acid 54548815 3,7,11,11-tetramethyldodeca-2,4-dienoicacid 545151057-methyl-3-[2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]nona-2,5-YLWKTERFWUXE dienoic acid BW-UHFFFAOYSA- N; 005B26AC36D10A0C9DB5EF006864943F 543589503-methyl-5-[2-[2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclohepten-1-yl]penta-2,4-dienoic acid 543537263,7,11,11-tetramethyltrideca-2,4-dienoic acid 541937133-methyl-5-[2-[2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cycloocten-1-yl]penta-2,4-dienoic acid 539467782,3,7-trimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 539448239-(6,6-dimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8- JAIGDKSXLVOFMtetraenoic acid H-UHFFFAOYSA- N; F42136BEED6C5A3 745B9BA23356D783053921377 3-methyl-5-[2-[2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclohexen-1-yl]penta-2,4-dienoic acid 44579100(2E,4E,6Z,8E)-9-[6,6-dimethyl-2-(2-methylpropyl)cyclohexen-1-CHEMBL476773 yl]-3,7-dimethylnona-2,4,6,8-tetraenoic acid 44579056(2E,4E,6E,8E)-9-[6,6-dimethyl-2-(2-methylpropyl)cyclohexen-1-CHEMBL476348 yl]-3,7-dimethylnona-2,4,6,8-tetraenoic acid 44314230(2Z,5E)-7-methyl-3-[(E)-2-(2,6,6-trimethylcyclohexen-1- CHEMBL75548;yl)ethenyl]nona-2,5-dienoic acid CHEBI:220121 25011742(2E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,8- dienoicacid 22646220 (2E,4E,6E,8E)-2,3-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 20830941(2E,4E,6E,8E)-2,3-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 19609240(2E,4E)-3-methyl-5-[(1Z)-2-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cycloocten-1-yl]penta-2,4-dienoic acid 18977383(2E,4E,6E,8E)-3,7-dimethyl-9-(2,5,6,6-tetramethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 15125883(2Z,4E,6E,8E)-2,3,7-trimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 15125882(2E,4E,6E,8E)-2,3,7-trimethyl-9-(2,6,6-trimethylcyclohexen-1-CHEMBL153895; yl)nona-2,4,6,8-tetraenoic acid 14-methyl-all-trans-retinoic acid; LMPR01090034 11266097(2Z,4E,8E)-3-methyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,8-trien-6-ynoic acid 11000660(2E,4E,6Z,8E)-9-(6,6-dimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid 10733921(2E,4E,6Z)-7-(8,8-dimethyl-4,5,6,7-tetrahydro-3H-naphthalen-2-yl)-3-methylocta-2,4,6-trienoic acid 10636975(2E,4E,6E,8E)-9-(6,6-dimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid 10591236(2E,4E,6Z)-7-(4a,8-dimethyl-4,5,6,7-tetrahydro-3H-naphthalen-2-yl)-3-methylocta-2,4,6-trienoic acid 10404132(Z,4E)-3-methyl-4-[(4E)-3-methyl-4-[(2,6,6-trimethylcyclohexen-1-yl)methylidene]cyclohex-2-en-1-ylidene]but-2-enoic acid 10314318(E,4E)-3-methyl-4-[(4E)-3-methyl-4-[(2,6,6-trimethylcyclohexen-1-yl)methylidene]cyclohex-2-en-1-ylidene]but-2-enoic acid 10215224(2E,4E,6Z,8E)-3-methyl-7-propan-2-yl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10193246(2E,4E)-3-methyl-6-[1-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclopropyl]hexa-2,4-dienoic acid 9841547(2E,4E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclohepten-1-yl]penta-2,4-dienoic acid 9830767(2Z,4E,6Z,8E)-9-(6,6-dimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid 9819335(2E,4E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclohexen-1- Ro 25-6603;173792- yl)ethenyl]cyclohexen-1-yl]penta-2,4-dienoic acid 73-9 56667667(2E,4E,6Z,8E)-3,7-dimethyl-9-(6-methyl-3-prop-1-en-2- CHEMBL455993;ylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid CHEMBL455994 54758572(2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-9-cis-Retinoate; yl)nona-2,4,6,8-tetraenoate CPD-13549 544266792,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8- tetraenoicacid 543251496-chloro-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 537026876-iodo-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 29986894(2E,4Z,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1- ZINC22066351yl)nona-2,4,6,8-tetraenoate 29927144(2E,4E,6E,8Z)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1- ZINC21992287yl)nona-2,4,6,8-tetraenoate 24916820(2E,4E,6E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona- 2g782,4,6-trienoate 247718173,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8- CHEBI:15036tetraenoate 21917290(2E,4E,6E,8E)-9-(5-tert-butyl-2,6,6-trimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid 19609245(2E,4E,6E,8E)-6-chloro-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 19609224(2E,4E,6E,8E)-6-iodo-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10924150(2E,4E,6Z,8E)-9-(2,6-dimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid 10613228(2E,4E,6E,8E)-9-(2,6-dimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid 10469989(2E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,6,8-trien-4-ynoic acid 10334998(2E,4E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclopropyl]penta-2,4-dienoic acid 9904356(2Z,4E,6Z)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6-trien-8-ynoic acid 7364357(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1- AC1OKKW8;yl)nona-2,4,6,8-tetraenoate ZINC12661824; 7048538(2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-13-cis-retinoate; yl)nona-2,4,6,8-tetraenoate ZINC03792789 64405652E,4E,6E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-7,8-Dehydroretinoic 2,4,6-trien-8-ynoic acid acid; 7,8-Didehydroretinoic acid 6419707(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1- Retinoate;all-trans- yl)nona-2,4,6,8-tetraenoate Retinoate; Tretinoine 5771658(Z)-3-(2,6,6-trimethylcyclohexen-1-yl)prop-2-enoic acid NSC-202789;AC1NY9IQ; NCGC00014560 5383969(E)-3-(2,6,6-trimethylcyclohexen-1-yl)prop-2-enoic acid NSC202789; NSC-20278 5353358 (2Z,4E)-3-methyl-6-(2,7,7-trimethyl-3-methylidene-1,4,5,6-AC1NS43Q tetrahydroinden-2-yl)hexa-2,4-dienoic acid 5289278(2E,4E)-3-methyl-6-[(2R)-2,7,7-trimethyl-3-methylidene-1,4,5,6-NSC202789; 3- tetrahydroinden-2-yl]hexa-2,4-dienoic acid(2,6,6-trimethyl-1- cyclohexen-1- yl)acrylic acid; AC1L77HZ 3057423-(2,6,6-trimethylcyclohexen-1-yl)prop-2-enoic acid NSC202789; 3-(2,6,6-trimethyl-1- cyclohexen-1- yl)acrylic acid; AC1L77HZ 18513-methyl-6-(2,7,7-trimethyl-3-methylidene-1,4,5,6- AC1L1CDOtetrahydroinden-2-yl)hexa-2,4-dienoic acid 543995426-bromo-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 542334763,7-dimethyl-5-oxo-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,6,8-trienoic acid 540331102,5,9-trimethyl-11-(2,6,6-trimethylcyclohexen-1-yl)undeca-2,4,6,8,10-pentaenoic acid 539367083-methyl-5-[2-[2-(2,6,6-trimethylcyclohexen-1-yl)ethynyl]cyclopenten-1-yl]penta-2,4-dienoic acid 44314320(2Z,4E)-3-methyl-5-[2-[(E)-2-(3,3,6,6-tetramethylcyclohexen-1-CHEMBL73973; yl)ethenyl]cyclopropyl]penta-2,4-dienoic acid CHEBI:22030344314319 (2E,4E)-3-methyl-5-[2-[(E)-2-(3,3,6,6-tetramethylcyclohexen-1-CHEMBL74331; yl)ethenyl]cyclopropyl]penta-2,4-dienoic acid CHEBI:22030122373193 (2E,4E)-3-methyl-5-[2-[2-(2,6,6-trimethylcyclohexen-1-yl)ethynyl]cyclopenten-1-yl]penta-2,4-dienoic acid 21145248(2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 20151571(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 19609231(2E,4E,6E,8E)-6-bromo-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 16727824(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-All-trans-Retinoic yl)nona-2,4,6,8-tetraenoic acid acid & 9-cis-RetinoicAcid 11015604(2E,4E,6E,8E,10E,12E,14E,16E)-2,6,11,15-tetramethyl-17-(2,6,6-trimethylcyclohexen-1-yl)-3-tritioheptadeca-2,4,6,8-trimethylcyclyhexen-1-yl)-3-tritioheptadeca-2,4,6,8,10,12,14,16-octaenoic acid 10406618(2E,4Z,6E,8E,10E,12E)-2,7,11-trimethyl-13-(2,6,6-trimethylcyclohexen-1-yl)trideca-2,4,6,8,10,12-hexanoic acid 9976193(2E,4E,6E,8E,10E,12E)-2,7,11-trimethyl-13-(2,6,6-trimethylcyclohexen-1-yl)trideca-2,4,6,8,10,12-hexanoic acid 9843074(2E,4E,6E)-3-methyl-7-(4,4,7,7-tetramethyl-2-pentyl-1,3,5,6-tetrahydroinden-2-yl)hepta-2,4,6-trienoic acid 6439881(2Z,4E,6Z,8E)-9-(3,3-difluoro-2,6,6-trimethylcyclohexen-1-yl)- DFRA;4,4- 3,7-dimethylnona-2,4,6,8-tetraenoic acid Difluororetinoic acid;AC1O5SM 6436320(2E,4E,6Z,8E,10E,12E,14E,16E)-2,6,11,15-tetramethyl-17-(2,6,6- AC1O5LFK;beta- trimethylcyclohexen-1-yl)heptadeca-2,4,6,8,10,12,14,16-octaenoicapo-8′-Carotenoic acid acid; 8′-Apo-beta,psi- carotenoic acid 5387557(2Z)-2-[5-(2,6,6-trimethylcyclohexen-1-yl)-3-[(E)-2-(2,6,6- NSC624510;trimethylcyclohexen-1-yl)ethenyl]cyclohexanoic acid AC1NTSHG; AC1Q5T6Y5366642 (2E,4E,6E,8E)-9-(3,3-difluoro-2,6,6-trimethylcyclohexen-1-yl)-4,4-Difluororetinoic 3,7-dimethylnona-2,4,6,8-tetraenoic acid acid;AC1NSNWF; 4,4-Difluororetinoic acid (all-trans) 3614732-[5-(2,6,6-trimethylcyclohexen-1-yl)-3-[2-(2,6,6- AC1L7IQC;trimethylcyclohexen-1-yl)ethenyl]cyclohex-2-en-1-yl)heptadeca-NCI60_007432; 2-[5- 2,4,6,8,10,12,14,16-octaenoic acid (2,6,6-trimethylcyclohexen- 1-yl)-3-[2-(2,6,6- trimethylcyclohexen- 1-yl)ethenyl]cyclohex- 2-en-1-ylidene]acetic acid 1462189-(3,3-difluoro-2,6,6-trimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid 56660872(2E,4E,6Z,8E)-3,7-dimethyl-9-(2-methyl-5-prop-1-en-2- CHEMBL457645;ylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid CHEMBL513434 54587023(2E,4E,6Z,8E)-3,7-dimethyl-9-[(3S,6R)-3-methyl-6-prop-1-en-2-CHEMBL1773351 ylcyclohexen-1-yl]nona-2,4,6,8-tetraenoic acid 54586043(2E,4E,6Z)-3-methyl-7-[(3R,6S)-3-methyl-6-propan-2- CHEMBL1773361ylcyclohexen-1-yl]octa-2,4,6-trienoic acid 543102027-ethyl-3,11-dimethyltrideca-2,4-dienoic acid 541779958-(3-ethyl-2-propan-2-ylcyclohex-2-en-1-ylidene)-3,7- OZUTXDDSOLQKNdimethylocta-2,4,6-trienoic acid K-UHFFFAOYSA- N; 982DADEA9DC5579A132BDF2AD7FA 647A 54012267 3,8,12-trimethyltrideca-2,4-dienoic acid53787191 3,8,13-trimethyltetradeca-2,4-dienoic acid 537431944-methyl-6-(2,6,6-trimethylcyclohexen-1-yl)hex-2-enoic acid 537105213,7,13-trimethyltetradeca-2,4-dienoic acid 537076703,7-dimethyl-8-(3-methyl-2-propan-2-ylcyclohex-2-en-1- BYHSFJNWVLBCIylidene)octa-2,4,6-trienoic acid M-UHFFFAOYSA- N; 14B10A34153F37A66327788679FAC42F 53666154 3,7,11-trimethyltrideca-2,4-dienoic acid53438161 3,7,11-trimethyltetradeca-2,4-dienoic acid 534277547,7-dimethylicosa-2,4-dienoic acid 52952998(2E,4E,6Z,8E)-3,7-dimethyl-9-[(3R,6S)-3-methyl-6-prop-1-en-2-CHEMBL1773352 ylcyclohexen-1-yl]nona-2,4,6,8-tetraenoic acid 44631433(2Z,4E)-3-methyl-5-(2,2,4-trimethylcyclohex-3-en-1-yl)penta-2,4-FZFFLFPGBIXCKI- dienoic acid STRRHFTISA- 44291210(2Z,4Z,6Z,8E)-8-(3-ethyl-2-propan-2-ylcyclohex-2-en-1-ylidene)-CHEMBL43954 3,7-dimethylocta-2,4,6-trienoic acid 44290946(2E,4Z,6Z,8E)-8-(3-ethyl-2-propan-2-ylcyclohex-2-en-1-ylidene)-CHEMBL43833; 3,7-dimethylocta-2,4,6-trienoic acid CHEBI:167938 24845989sodium (2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6- LS-143475trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoate 23670222 potassium(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoate 23665641 sodium(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6- Sodium retinoate;trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoate Retinoic acid, sodiumsalt; Vitamin A acid sodium sal 23265304(2E,4E)-3-methyl-5-(2,2,4-trimethylcyclohex-3-en-1-yl)penta-2,4- dienoicacid 21437585 (2E,4E)-3,8,12-trimethyltrideca-2,4-dienoic acid 21437539(2E,4E)-3,8,13-trimethyltetradeca-2,4-dienoic acid 21437504(2E,4E)-3,7,13-trimethyltetradeca-2,4-dienoic acid 21158960(2E,4E)-7,7-dimethylicosa-2,4-dienoic acid 20270951(6E,8E)-2,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,3,6,8-tetraenoic acid 19609232(2E,4E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclohexen-1-yl]penta-2,4-dienoic acid 11130378(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-2-en-1-yl)nona-2,4,6,8-tetraenoic acid 11066537(2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-2-en-1-yl)nona-2,4,6,8-tetraenoic acid 10470917(2Z,4E,6Z,8E)-8-(3-ethyl-2-propan-2-ylcyclohex-2-en-1-ylidene)-3,7-dimethylocta-2,4,6-trienoic acid 10402558(2Z,4E,6E,8E)-3,7-dimethyl-8-(3-methyl-2-propan-2-ylcyclohex-2-en-1-ylidene)octa-2,4,6-trienoic acid 10357464(2E,4E,6Z,8E)-3,7-dimethyl-8-(3-methyl-2-propan-2-ylcyclohex-2-en-1-ylidene)octa-2,4,6-trienoic acid 10086191(2E,4E,6E,8E)-3,7-dimethyl-8-(3-methyl-2-propan-2-ylcyclohex-CHEMBL333032; 2-en-1-ylidene)octa-2,4,6-trienoic acid CHEBI:29941010086189 (2Z,4E,6Z,8E)-3,7-dimethyl-8-(3-methyl-2-propan-2-ylcyclohex-2-en-1-ylidene)octa-2,4,6-trienoic acid 9972952(2Z,4E,6E,8E)-8-(3-ethyl-2-propan-2-ylcyclohex-2-en-1-ylidene)-CHEMBL44582; 3,7-dimethylocta-2,4,6-trienoic acid CHEBI:168408 9972949(2E,4E,6Z,8E)-8-(3-ethyl-2-propan-2-ylcyclohex-2-en-1-ylidene)-3,7-dimethylocta-2,4,6-trienoic acid 9883342(2E,4E,6E,8E)-8-(3-ethyl-2-propan-2-ylcyclohex-2-en-1-ylidene)-CHEMBL46398; 3,7-dimethylocta-2,4,6-trienoic acid CHEBI:168441 5372326(E)-3-methyl-5-(2,6,6-trimethylcyclohexen-1-yl)pent-2-enoic acidAC1NSY3I; 2- Pentenoic acid, 3- methyl-5-(2,6,6- trimethyl-1-cyclohexenyl); (E)-3- methyl-5-(2,6,6- trimethylcyclohexen-1-yl)pent-2-enoic acid 445560(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-2-en-1- AC1L9I79yl)nona-2,4,6,8-tetraenoic acid 56667221(2E,4E,6Z,8E)-3,7-dimethyl-9-(3-methyl-6-propan-2- CHEMBL508378ylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 54585066(2E,4E,6Z,8E)-3,7-dimethyl-9-[(1S,4R,5R)-4,6,6-trimethyl-3-CHEMBL1773358 bicyclo[3.1.1]hept-2-enyl]nona-2,4,6,8-tetraenoic acid54585064 (2E,4E,6Z,8E)-3,7-dimethyl-9-[(3R)-3-methyl-6-propan-2-CHEMBL1773355 ylidenecyclohexen-1-yl]nona-2,4,6,8-tetraenoic acid54582176 (2E,4E,6Z,8E)-3,7-dimethyl-9-[(3S)-3-methyl-6-propan-2-CHEMBL1773354 ylidenecyclohexen-1-yl]nona-2,4,6,8-tetraenoic acid54581148 (2E,4E,6Z,8E)-3,7-dimethyl-9-[(1R,2R,5S)-2-methyl-5-propan-2-CHEMBL1773360 yl-3-bicyclo[3.1.0]hex-3-enyl]nona-2,4,6, 8-tetraenoicacid 54542310 3,4,4-trimethyltetradec-2-enoic acid 545210543,4,4-trimethyloctadec-2-enoic acid 545186733,7-dimethyl-9-(2,6,6-trimethyl-5-oxocyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 54348687 3,7,10,11-tetramethyldodeca-2,4-dienoicacid 54325421 3,4,4-trimethylheptadec-2-enoic acid 543164933,4,4-trimethylpentadec-2-enoic acid 543050442-ethyl-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 542656803,7,11,15-tetramethylhexadeca-2,4-dienoic acid 541943593,7-dimethyl-9-(2,6,6-trimethyl-4-oxocyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 541704673,7,11,15-tetramethylhexadeca-2,4,6,14-tetraenoic acid 541671723,4,4-trimethylhexadec-2-enoic acid 541058653,7,7,11,11-pentamethyldodec-2-enoic acid 540642532-ethyl-5,9-dimethyl-3-(2,6,6-trimethylcyclohexen-1-yl)undeca-2,4,6,8,10-pentaenoic acid 539613713,7,11-trimethyldodeca-2,4,11-trienoic acid 539366029-[5-(2-cyclohexylethyl)-2,6,6-trimethylcyclohexen-1-yl]-3,7-dimethylnona-2,4,6,8-tetraenoic acid 538252333,7,11,15,19-pentamethylicosa-2,4,6,10,18-pentaenoic acid 538015693-methyl-5-[2-[2-(2,6,6-trimethylcyclohexen-1-yl)ethynyl]cyclohepten-1-yl]penta-2,4-dienoic acid 537258053,7-dimethyldodeca-2,4-dienoic acid 537004163,7,11,15-tetramethylhexadeca-2,4,6-trienoic acid 52953080(2E,4E,6Z,8E)-3,7-dimethyl-9-[(3S,6R)-3-methyl-6-propan-2- CHEMBL1773353ylcyclohexen-1-yl]nona-2,4,6,8-tetraenoic acid 52952997(2E,4E,6Z,8E)-3,7-dimethyl-9-[(1R,4S,5S)-4,6,6-trimethyl-3-CHEMBL1773357 bicyclo[3.1.1]hept-2-enyl]nona-2,4,6,8-tetraenoic acid52921782 (2E,5R,10E,12E)-3,5,15-trimethyl-7-methylidenehexadeca-LMFA01020367; 2,10,12-trienoic acid 16:3(2E,10E,12E)(3Me,5Me[R],7My,15Me) 46178652(2E,4E)-5-[(1R)-2,2-dimethyl-6-methylidenecyclohexyl]-3-methylpenta-2,4-dienoic acid 44579059(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,2,6-trimethylcyclohexyl)nona-CHEMBL451158 2,4,6,8-tetraenoic acid 25147656(2E,4E,6Z,8E)-3,7-dimethyl-9-[(3R,6S)-3-methyl-6-propan-2- CHEMBL508378ylcyclohexen-1-yl]nona-2,4,6,8-tetraenoic acid 22168242(2E,4E,6E,10E)-3,7,11,15,19-pentamethylicosa-2,4,6,10,18- pentaenoicacid 22168239 (2E,4E,6E)-3,7,11,15-tetramethylhexadeca-2,4,6-trienoicacid 22168234(2E,4E,6E)-3,7,11,15-tetramethylhexadeca-2,4,6,14-tetraenoic acid21764469 (2E,4E)-3-methyl-5-[(1R)-2,6,6-trimethylcyclohex-2-en-1-yl]penta-2,4-dienoic acid 21650797 acetyl(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraeneperoxoate 21525820(2E,4E)-7,11,11-trimethyldodeca-2,4-dienoic acid 21525806(2E,4E)-3,7-dimethyldodeca-2,4-dienoic acid 21291068(E)-3,4,4-trimethylhexadec-2-enoic acid 21291063(E)-3,4,4-trimethyltetradec-2-enoic acid 21291060(E)-3,4,4-trimethylpentadec-2-enoic acid 21291047(E)-3,4,4-trimethylheptadec-2-enoic acid 21291045(E)-3,4,4-trimethyloctadec-2-enoic acid 20830940(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,5,6,6-tetramethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoate 20306860(2E,4E)-3,7,11-trimethyldodeca-2,4,11-trienoic acid 20027300 azanium(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoate 19609235(2E,4E)-2-iodo-3-methyl-5-(2,6,6-trimethylcyclohexen-1-yl)penta-2,4-dienoic acid 19606927(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-4-oxocyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 18977382(2E,4E,6E,8E)-3,7-dimethyl-9-(2,5,6,6-tetramethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoate 16061319(2Z,4E,6Z,8E)-7-(hydroxymethyl)-3-methyl-9-(2,6,6- 19-Hydroxy-13-cis-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid retinoic acid;LMPR01090029 16061318 (2E,4E,6Z,8E)-7-(hydroxymethyl)-3-methyl-9-(2,6,6-19-Hydroxy-all-trans- trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoicacid retinoic acid; LMPR01090028 15125888(2E,4E,6E,8E)-2-ethyl-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-CHEMBL154239 1-yl)nona-2,4,6,8-tetraenoic acid 11747707(2E,4E,6Z,8E)-3,7-dimethyl-9-(6-methylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 11602784(2E,4E)-3-methyl-5-[2-[2-(2,6,6-trimethylcyclohexen-1-yl)ethynyl]cyclohepten-1-yl]penta-2,4-dienoic acid 10516342(2E,4E,6E,8E)-3,7-dimethyl-9-(6-methylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 10354668(Z,4E)-4-(3-ethyl-2-propan-2-ylcyclohex-2-en-1-ylidene)-3-methylbut-2-enoic acid 10053647(2Z,4Z,6E,8E,10E,12E,14E,16E,18E,20E,22E,24E)-2,6,10,14,19,23-hexamethyl-25-(2,6,6-trimethylcyclohexen-1-yl)pentacosa-2,4,6,8,10, 12,14,16,18,20,22,24-dodecaenoic acid 9995780(2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-5-oxocyclohexen-Oxo-13-cis-retinoate; 1-yl)nona-2,4,6,8-tetraenoic acid 4-keto-13-cis-retinoate 9949957(2E,4E,6E,8E)-3,7-dimethyl-8-[3-(2-methylpropyl)-2-propan-2-ylcyclohex-2-en-1-ylidene]octa-2,4,6-trienoic acid 9948768(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-5-oxocyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 9829386(2E,4Z,6E,8E,10E,12E,14E,16E,18E,20E,22E,24E)-2,6,10,14,19,23-hexamethyl-25-(2,6,6-trimethylcyclohexen-1-yl)pentacosa-2,4,6,8,10, 12,14,16,18,20,22,24-dodecaenoic acid 6477090(2Z,4Z,6Z,8E,10Z,12Z,14E,16Z,18Z,20E,22Z,24E)- AC1O53P5; 3′,4′-2,6,10,14,19,23-hexamethyl-25-(2,6,6-trimethylcyclohexen-1-Didehydro-,.psi.- yl)pentacosa-2,4,6,8,10, caroten-16′-oic acid12,14,16,18,20,22,24-dodecaenoic acid 6439734(2Z,4E,6Z,8E)-3,7-dimethyl-9-(2,2,6-trimethylcyclohexyl)nona-7,8-Dihydroretinoic 2,4,6,8-tetraenoic acid acid 6437018(2Z,4E)-3,7,11-trimethyldodeca-2,4-dienoic acid AC1O5MUO; EINECS258-354-9 6437016 (2E,4E)-3,7,11-trimethyldodeca-2,4-dienoic acidAC1O5MUI; CHEMBL37590 5476505(2E,4E)-3-methyl-5-(2,6,6-trimethylcyclohex-2-en-1-yl)penta-2,4-AC1O5MUI; dienoic acid CHEMBL37590 5460164(2E,4E,6E,8E)-3,7-dimethyl-9-(2,2,6-trimethylcyclohexyl)nona- Retinylester; all- 2,4,6,8-tetraenoic acid trans-Retinyl ester 5281248(2E,4E,6E,8E,10E,12E,14E,16E,18E,20E,22E,24E)- NSC635690;2,6,10,14,19,23-hexamethyl-25-(2,6,6-trimethylcyclohexanoicTorularhodin; acid AC1NQY9 6370392E,4E,6E,8E,10E,12E,14E,16E,18E,20E)-2,6,10,15,19- Neurosporaxanthin;pentamethyl-21-(2,6,6-trimethylcyclohexen-1-yl)hexanoic acid all-trans-Neurosporaxanthin 4284853-methyl-5-(2,6,6-trimethylcyclohex-2-en-1-yl)penta-2,4-dienoicAC1L8LML; 3- acid methyl-5-(2,6,6- trimethylcyclohex-2-en-1-yl)penta-2,4- dienoic acid 1037233,7,11-trimethyldodeca-2,4-dienoic acid 941652,6,10,14,19,23-hexamethyl-25-(2,6,6-trimethylcyclohexen-1- AC1L3RN8;yl)pentacosa-2,4,6,8,10,12,14,16,18,20,22,24-dodecanenoic acidNCI60_011910 56661049(2E,4E,6Z,8E)-3,7-dimethyl-9-(4,4,6,6-tetramethyl-2- CHEMBL455992bicyclo[3.1.1]hept-2-enyl)nona-2,4,6,8-tetraenoic acid 54581147(2E,4E,6Z,8E)-9-[(1S,5R)-6,6-dimethyl-4-bicyclo[3.1.1]hept-3-CHEMBL1773359 enyl]-3,7-dimethylnona-2,4,6,8-tetraenoic acid 544780243,4,4-trimethylnon-2-enoic acid 54476971 3,4,4-trimethylundec-2-enoicacid 54287870 3-formyl-7-methyl-9-(2,6,6-trimethyleyclohexen-1-yl)nona-RVKZSGIKOAAYJJ- 2,4,6,8-tetraenoic acid UHFFFAOYSA-N; 293564D2B64FAC5F524A1B691CBF7C 6B 541163973,7-dimethyl-2-propan-2-yl-9-(2,6,6-trimethylcyclohexen-1-NKQIYDSGIYJXSA- yl)nona-2,4,6,8-tetraenoic acid UHFFFAOYSA-N;5597749F477D668D 55E163C44DA1F3EB 54073647 3,4,4-trimethyldec-2-enoicacid 53995964 3-methyl-5-[2-[2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclohexyl]penta-2,4-dienoic acid 539197983,4,4-trimethyldodec-2-enoic acid 538899223,7-dimethyl-9-(2,4,4,6,6-pentamethyl-3-oxocyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 538874604-(hydroxymethyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 538547963-methyl-6-(3,3,7,7-tetramethyl-3a,4,5,6-tetrahydroinden-2-ylidene)hexa-2,4-dienoic acid 537546092-ethyl-5,9-dimethyl-11-(2,6,6-trimethylcyclohexen-1-yl)undeca-2,4,6,8,10-pentaenoic acid 50925583(2E,4E,6E,8E)-9-[(1R,2R,4aS,8aR)-1,6-dimethyl-2-propyl-4a,5,8,8a-tetrahydro-2H-naphthalen-1-yl]-8-methylnona-2,4,6,8-tetraenoic acid 45039634(2E,4E,6E,8E)-9-[6,6-dimethyl-3-oxo-2-(trideuteriomethyl)cyclohcxen-1-yl]-3,7-dimethylnona-2,4,6,8- tetraenoicacid 21291081 (E)-3,4,4-trimethyldec-2-enoic acid 21291044(E)-3,4,4-trimethyldodec-2-enoic acid 21291042(E)-3,4,4-trimethylnon-2-enoic acid 21291032(E)-3,4,4-trimethylundec-2-enoic acid 19384872(E)-4-[(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoyl]oxy-4-oxobut-2-enoic acid 16061321(2Z,4E,6Z,8E)-7-formyl-3-methyl-9-(2,6,6-trimethylcyclohexen-19-Oxo-9-cis-retinoic 1-yl)nona-2,4,6,8-tetraenoic acid acid;LMPR01090031 16061320(2E,4E,6Z,8E)-7-formyl-3-methyl-9-(2,6,6-trimethylcyclohexen-19-Oxo-all-trans- 1-yl)nona-2,4,6,8-tetraenoic acid retinoic acid;LMPR01090030 15125894 (2E,4E,6E,8E)-3,7-dimethyl-2-propan-2-yl-9-(2,6,6-CHEMBL153894 trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid10043037 (2E,4E,6E,8E)-3,7-dimethyl-9-(2,4,4,6,6-pentamethyl-3-CHEMBL103068 oxocyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 9972939(2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-3-oxocyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 9906064(2E,4E)-3-methyl-5-[(1R)-2-[(E)-2-(2,6,6-trimethylcyclohexen-1-yl)ethenyl]cyclohexyl]penta-2,4-dienoic acid 9902057(2Z,4E,6Z,8E)-4-(hydroxymethyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid 6437087(2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-3-oxocyclohexen-Oxoretinoic acid; 4- 1-yl)nona-2,4,6,8-tetraenoic acid Oxo-isotretinoin6437063 (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethyl-3-oxocyclohexen-4-Oxoretinoic acid; 1-yl)nona-2,4,6,8-tetraenoic acid 4-Ketoretinoicacid 447276(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexa-1,3-dien- VitaminA2 acid; 3,4- 1-yl)nona-2,4,6,8-tetraenoic acid Didehydroretinoic acid104857 3,7-dimethyl-9-(2,6,6-trimethyl-3-oxocyclohexen-1-yl)nona-2,4,6,8-tetraenoic acid

The term “aryl,” as used herein, represents a mono- or bicyclic C₆-C₁₄group with [4n+2]π electrons in conjugation and where n is 1, 2, or 3.Aryl groups also include ring systems where the ring system having[4n+2]π electrons is fused to a non-aromatic cycloalkyl or anon-aromatic heterocyclyl. Phenyl is an aryl group where n is 1. Arylgroups may be unsubstituted or substituted with, e.g., 1, 2, 3, or 4substituent groups as defined herein. Still other exemplary aryl groupsinclude, but are not limited to, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, and indenyl.

The term “cycloalkyl,” as used herein, represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to tencarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl,and the like. In some embodiments, the cycloalkyl is a polycyclic (e.g.,adamantyl). Cycloalkyl groups may be unsubstituted or substituted with,e.g., 1, 2, 3, or 4 substituent groups as defined herein.

The term “heteroaryl,” as used herein, represents an aromatic (i.e.,containing 4n+2 pi electrons within the ring system) 5- or 6-memberedring containing one, two, three, or four heteroatoms independentlyselected from the group consisting of nitrogen, oxygen, and sulfur, aswell as bicyclic, tricyclic, and tetracyclie groups in which any of thearomatic ring is fused to one, two, or three heterocyclic or carbocyclicrings (e.g., an aryl ring). Exemplary heteroaryls include, but are notlimited to, furan, thiophene, pyrrole, thiadiazole (e.g.,1,2,3-thiadiazole or 1,2,4-thiadiazole), oxadiazole (e.g.,1,2,3-oxadiazole or 1,2,5-oxadiazole), oxazole, isoxazole, isothiazole,pyrazole, thiazole, triazole (e.g., 1,2,4-triazole or 1,2,3-triazole),pyridine, pyrimidine, pyrazine, pyrazine, triazine (e.g., 1,2,3-triazine1,2,4-triazine, or 1,3,5-triazine), 1,2,4,5-tetrazine, indolyl,quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, andbenzoxazolyl. Heteroaryls may be unsubstituted or substituted with,e.g., 1, 2, 3, or 4 substituents groups as defined herein.

The term “heterocyclyl,” as used herein represents a non-aromatic 5-, 6-or 7-membered ring, unless otherwise specified, containing one, two,three, or four heteroatoms independently selected from the groupconsisting of nitrogen; oxygen, and sulfur. Heterocyclyl groups may beunsubstituted or substituted with, e.g., 1, 2, 3, or 4 substituentgroups as defined herein.

Where a group is substituted, the group may be substituted with 1, 2, 3,4, 5, or 6 substituent groups. Optional substituent groups include, butare not limited to: C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,heteroaryl, cycloalkyl, heterocyclyl, halogen (—F, —Cl, —Br, or —I),azido(—N₃), nitro (—NO₂), cyano (—CN), acyloxy(—OC(═O)R′), acyl(—C(═O)R′), alkoxy (—OR′), amido (—NR′C(═O)R″ or —C(═O)NRR′), amino(—NRR′), carboxylic acid (—CO₂H), carboxylic ester (—CO₂R′), carbamoyl(—OC(═O)NR′R″ or —NRC(═O)OR′), hydroxy (—OH), oxo (═O), isocyano (—NC),sulfonate (—S(═O)₂OR), sulfonamide (—S(═O)₂NRR′ or —NRS(═O)₂R′), orsulfonyl (—S(═O)₂R), where each R or R′ is selected, independently, fromH, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl, or heteroaryl. In someembodiments, the substituent groups themselves may be furthersubstituted with, for example, 1, 2, 3, 4, 5, or 6 substituents asdefined herein. For example, a C₁₋₆ alkyl, aryl, or heteroaryl group maybe further substituted with 1, 2, 3, 4, 5, or 6 substituents asdescribed herein.

The retinoic acid compounds of the invention inhibit Pin1 activity(e.g., as determined by the fluorescence polarization-based displacementassay or PPIase assay as describe herein). This inhibition can be, e.g.,greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, orgreater.

The language “anti-proliferative compound” is intended to includechemical reagents which inhibit the growth of proliferating cells ortissues wherein the growth of such cells or tissues is undesirable.Chemotherapeutic agents are well known in the art (as well as describedherein), and are typically used to treat neoplastic diseases, tumors,and cancers.

“Treatment,” as used herein, is defined as the application oradministration of a therapeutic agent (e.g., a retinoic acid compound)to a patient, or application or administration of a therapeutic agent toan isolated tissue or cell line from a patient, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease, or to slow the progression of thedisease.

As used herein, the terms “sample” and “biological sample” includesamples obtained from a mammal or a subject containing Pin1 which can beused within the methods described herein, e.g., tissues, cells andbiological fluids isolated from a subject, as well as tissues, cells andfluids present within a subject. Typical samples from a subject includetissue samples, tumor samples, blood, urine, biopsies, lymph, saliva,phlegm, pus, and the like.

By a “low dosage” or “low concentration” is meant at least 5% less(e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) than the loweststandard recommended dosage or lowest standard recommended concentrationof a particular compound formulated for a given route of administrationfor treatment of any human disease or condition. For example, a lowdosage of an anti-proliferative compound formulated for oraladministration will differ from a low dosage of an anti-proliferativecompound formulated for intravenous administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic showing relationship between Pin1 and theactivity of the indicated genes.

FIG. 1B is a schematic showing the role of Pin1 in the indicated signaltransduction pathways.

FIG. 2A is a schematic of the chemical structure of 13-cis-retinoic acidand all-trans retinoic acid.

FIG. 2B is a schematic showing additional retinoic acid compounds.

FIG. 3 is a pair of graphs showing the ability of 13-cis-retinoic acidand all-trans retinoic acid at different concentrations to compete witha fluorescence-labeled Pin1 peptide inhibitor probe BIKL-488 for bindingPin1 after an incubation of the indicated amount of time.

FIG. 4 is a pair of graphs showing inhibition of Pin1 by cis-retinoicacid and trans-retinoic acid at the indicated concentrations.

FIG. 5 is a photograph indicating both cis and trans retinoic acids canbind to Pin1 and compete away DAPK, which binds to the Pin1 catalyticdomain.

FIG. 6A is a graph showing specific residues in the Pin1 active sitethat are important for interacting with the detecting probe BIKL-488.

FIG. 6B is a graph showing specific residues in the Pin1 active sitethat are important for interacting with retinoic acid.

FIG. 7A is a pair of graphs showing cell viability of the indicated celllines as a function of concentration of cis-retinoic acid ortrans-retinoic acid.

FIG. 7B is a series Western blots showing expression of the indicatedprotein in cells treated with of either cis-retinoic acid ortrans-retinoic acid in the indicated cell lines.

FIG. 8A is a photograph indicating the amount of Pin1 or GAPDH mRNAproduced in the presence of the indicated compound as measured in anRT-PCR experiment.

FIG. 8B is a Western blot showing Pin1 and tubulin protein levels incells treated with the indicated compounds

FIG. 8C is a Western blot showing Pin1 and tubulin protein levels incells treated with cycloheximide and the indicated compound.

FIG. 9A is a pair of graphs showing cell viability as a function of theconcentration of all trans-retinoic acid in the indicated cell types.

FIG. 9B is a Western blot showing Pin1 protein levels in the indicatedcell types.

FIG. 9C is Western blot showing Pin1 protein levels in the indicatedcell types treated with either 13cRA or ATRA.

FIG. 10A is a series of schematics showing the indicated retinoic acidcompounds and β-carotene.

FIG. 10B is a graph showing the concentration of free particles of Pin1as a function of concentration of the indicated compounds.

FIG. 10C is a table summarizing the results of FIG. 10B.

FIG. 10D is a series of schematics showing compounds that modulate theretinoic acid receptor.

FIG. 11A is a schematic of the chemical structure of all-trans retinoicacid and acitretin.

FIG. 11B is a graph showing the concentration of free particles of Pin1as a function of concentration of the indicated compounds.

FIG. 11C is a Western blot showing Pin1 and tubulin protein levels incells treated with the different concentrations of the indicatedcompound.

FIG. 11D is a graph showing cell viability as a function of theconcentration of the indicated compounds.

FIG. 12 is a graph showing cell number as a function of theconcentration of the indicated compound or combination of compounds. Fordata points corresponding to the combination of compounds, theconcentration value corresponds to the concentration of each individualcompound in the combination.

FIG. 13A is a Western blot showing Pin, Her2, and actin protein levelsin human breast cancer cells treated with the siRNA to inhibit Pin1.

FIG. 13B is a graph showing cell number as a function of inhibiting Pin1in cells by siRNA.

DETAILED DESCRIPTION OF THE INVENTION

In general, the invention features methods of treating a proliferativedisorder characterized by elevated Pin1 marker levels in a subject byadministering a retinoic acid compound. Additionally, the inventionfeatures methods of treating proliferative disorders (e.g.,proliferative disorders characterized by elevated Pin1 marker levels) byadministering a retinoic acid compound in combination with one or moreadditional anti-proliferative compounds or other anti-cancer therapies.

Inhibitors of Pin1 (e.g., retinoic acid compounds) are useful fortreating proliferative disorders (e.g., disorders characterized byincreased Pin1 activity). Furthermore, because Pin1 acts in severaldifferent oncogenic pathways, Pin1 inhibition would be expected tobehave synergistically with many anti-proliferative compounds

I. Pin1

Phosphorylation on serine/threonine-proline motifs restrains cis/transprolyl isomerization, and also creates a binding site for the essentialprotein Pin1. Pin1 binds and regulates the activity of a defined subsetof phosphoproteins, as well as participating in the timing of mitoticprogression. Both structural and functional analyses have indicated thatPin1 contains a phosphoserine/threonine-binding module that bindsphosphoproteins, and a catalytic activity that specifically isomerizesthe phosphorylated phosphoserinelthreonine-proline. Both of these Pin1activities are essential for Pin1 to carry out its function in vivo.

Pin1 is overexpressed in human cancer samples and the levels of Pin1 arecorrelated with the aggressiveness of tumors. We have found that apotent anticancer reagent with an unknown mechanism potently andreversibly inhibits Pin1 isomerase activity. Moreover, inhibition ofPin1 by various approaches, including the Pin1 inhibitor, Pin1 antisensepolynucleotides, or genetic depletion, kills human and yeast dividingcells by inducing premature mitotic entry and apoptosis. Thus, uponphosphorylation, Pin1 latches onto phosphoproteins and twists thepeptide bond next to the proline, which regulates the function ofphosphoproteins and participates in controlling the timing of mitoticprogression.

Pin1 is highly conserved and contains a protein-interacting module,called WW domain, and a catalytically active peptidyl-prolyl isomerase(PPIase). Pin1 is structurally and functionally distinct from members oftwo other well-characterized families of PPIases, the cyclophilins andthe FKBPs. PPIases are ubiquitous enzymes that catalyze the typicallyslow prolyl isomerization of proteins, allowing relaxation of localenergetically unfavorable conformational states. Phosphorylation onSer/Thr residues immediately preceding Pro not only alters the prolylisomerization rate, but also creates a binding site for the WW domain ofPin1. The WW domain acts a novel phosphoserine-binding module targetingPin1 to a highly conserved subset of phosphoproteins. Furthermore, Pin1displays a unique phosphorylation-dependent PPIase that specificallyisomerizes phosphorylated Ser/Thr-Pro bonds and regulates the functionof phosphoproteins.

Taken together, these results indicate that the Pin1 subfamily ofenzymes is a diagnostic and therapeutic target for diseasescharacterized by uncontrolled cell proliferation, primarilymalignancies.

II. Measurement of PIN1 Marker Levels

The present invention pertains to the treatment of proliferativediseases identified as coinciding with elevated Pin1 marker levels withretinoic acid compounds. In some aspects, the invention features thedetermination of Pin1 marker levels in a subject; where retinoic acid isadministered in subjects where Pin1 marker levels are determined to beelevated. In other aspects, the invention can also feature themeasurement of Pin1 marker levels subsequent to the administration ofretinoic acid compounds in order to evaluate the progress of therapy intreating the proliferative disorder.

Accordingly, one aspect of the present invention relates to diagnosticassays for measuring levels of Pin1 marker, as well as Pin1 activity, inthe context of a biological sample (e.g., tumor samples, blood, urine,biopsies, lymph, saliva, phlegm, and pus) to thereby determine whetheran individual is a candidate for treatment with a retinoic acidcompound. The invention features both treatment of subjects exhibitingsymptoms of a proliferative disorder and individuals at risk fordeveloping a proliferative disorder.

Diagnostic Assays

An exemplary method for detecting the presence or absence of Pin1protein or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting Pin1 protein ora nucleic acid (e.g., mRNA, genomic DNA) that encodes Pin1 protein suchthat the presence of Pin1 protein or nucleic acid is detected in thebiological sample. A preferred agent for detecting Pin1 mRNA or genomicDNA is a labeled nucleic acid probe capable of hybridizing to Pin1 mRNAor DNA. The nucleic acid probe can be, for example, a Pin1 nucleic acidor a corresponding nucleic acid such as an oligonucleotide of at least15, 30, 50, 100, 250 or 500 nucleotides in length which is capable ofspecifically hybridizing under stringent conditions to Pin1 mRNA orgenomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.

A preferred agent for detecting Pin1 marker is an antibody capable ofbinding to Pin1 protein, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. Theterm “labeled,” with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin.

With respect to antibody-based detection techniques, one of skill in theart can raise anti-Pin1 antibodies against an appropriate immunogen,such as isolated and/or recombinant Pin1 or a portion or fragmentthereof (including synthetic molecules, such as synthetic peptides)using no more than routine experimentation. Synthetic peptides can bedesigned and used to immunize animals, such as rabbits and mice, forantibody production. The nucleic and amino acid sequence of Pin1 isknown (Hunter et al., WO 97/17986 (1997); Hunter et al., U.S. Pat. Nos.5,952,467 and 5,972,697, the teachings of all of which are herebyincorporated by reference in their entirety) and can be used to designnucleic acid constructs for producing proteins for immunization or innucleic acid detection methods or for the synthesis of peptides forimmunization.

Conditions for incubating an antibody with a test sample can varydepending upon the tissue or cellular type. Incubation conditions candepend on the format employed in the assay, the detection methodsemployed, and the type and nature of the antibody used in the assay. Oneskilled in the art will recognize that any one of the commonly availableimmunological assay formats (such as radioimmunoassays, enzyme-linkedimmunosorbent assays, diffusion based Ouchterlony, or rocketimmunofluorescent assays) can readily be adapted to employ theantibodies of the present invention. Examples of such assays can befound in Chard, “An Introduction to Radioimmunoassay and RelatedTechniques,” Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock et al., “Techniques in Immunocytochemistry,” AcademicPress, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985);Tijssen, “Practice and Theory of enzyme Immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology,” is Elsevier SciencePublishers, Amsterdam, The Netherlands (1985).

The detection method of the invention can be used to detect Pin1 mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of Pin1 mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of Pin1 protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations,immunofluorescence, or quantitative sequencing reactions. In vitrotechniques for detection of Pin1 genomic DNA include Southernhybridizations. The detection of genomic mutations in Pin1 (or othergenes that effect Pin1 marker levels) can be used to identify inheritedor somatic mutations. Furthermore, in vivo techniques for detection ofPin1 protein include introducing into a subject a labeled anti-Pin1antibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

In another embodiment, the biological sample contains protein moleculesfrom the test subject. Alternatively, the biological sample can containmRNA molecules from the test subject or genomic DNA molecules from thetest subject. A preferred biological sample is a serum sample isolatedby conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting Pin1 marker such that thepresence of Pin1 marker is detected in the biological sample, andcomparing the presence of Pin1 marker in the control sample with thepresence of Pin1 marker in the test sample.

The immunological assay test samples of the present invention mayinclude cells, protein or membrane extracts of cells, blood orbiological fluids such as ascites fluid or brain fluid (e.g.,cerebrospinal fluid). The test sample used in the above-described methodis based on the assay format, nature of the detection method and thetissues, cells or extracts used as the sample to be assayed. Methods forpreparing protein extracts or membrane extracts of cells are well knownin the art and can be readily be adapted in order to obtain a samplewhich is capable with the system utilized. The invention alsoencompasses kits for detecting the presence of Pin1 in a biologicalsample. For example, the kit can comprise a labeled compound or agentcapable of detecting Pin1 protein or mRNA in a biological sample; meansfor determining the amount of Pin1 in the sample; and means forcomparing the amount of Pin1 in the sample with a standard. The compoundor agent can be packaged in a suitable container. The kit can furthercomprise instructions for using the kit to detect Pin1 protein ornucleic acid.

Pin1 marker levels can also be measured in an assay designed to evaluatea panel of target genes, e.g., a microarray or multiplex sequencingreaction. In the embodiments of the invention described herein, wellknown biomolecular methods such as northern blot analysis, RNaseprotection assays, southern blot analysis, western blot analysis, insitu hybridization, immunocytochemical procedures of tissue sections orcellular spreads, and nucleic acid amplification reactions (e.g.,polymerase chain reactions) may be used interchangeably. One of skill inthe art would be capable of performing these well-established protocolsfor the methods of the invention. (See, for example, Ausubel, et al.,“Current Protocols in Molecular Biology,” John Wiley & Sons, NY, N.Y.(1999)).

Diagnostic assays can be carried out in, e.g., subjects diagnosed or atrisk of a proliferative disorder. Such disorders include, withoutlimitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia,acute myelocytic leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acute monocyticleukemia, acute erythroleukemia, chronic leukemia, chronic myelocyticleukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma(Hodgkin's disease, non-Hodgkin's disease), Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors such assarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).

Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant Pin1 expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with Pin1 marker (e.g., aproliferative disorder). Thus, the present invention provides a methodfor identifying a disease or disorder associated with aberrant Pin1expression or activity in which a test sample is obtained from a subjectand Pin1 protein or nucleic acid (e.g., mRNA, genomic DNA) is detected,wherein the presence of Pin1 protein or nucleic acid is diagnostic for asubject having or at risk of developing a Pin1-associated disorder andare, therefore, susceptible to treatment with a retinoic acid compound.

Furthermore, the present invention provides methods for determiningwhether a subject can be effectively treated with a retinoic acidcompound for a disorder associated with aberrant Pin1 expression oractivity in which a test sample is obtained and Pin1 protein or nucleicacid expression or activity is detected (e.g., wherein the abundance ofPin1 protein or nucleic acid expression or activity is diagnostic for asubject that can be administered the agent to treat a disorderPin1-associated disorder).

In one embodiment, the present invention provides methods fordetermining Pin1 post-translational modifications. For example,phosphorylation of Pin1 on Ser71 in the catalytic active site by thetumor suppressor DAPK1 completely inhibits Pin1 catalytic activity andcell function to promote oncogenesis. More importantly, phosphorylationof Pin1 on Ser71 in the catalytic active site also prevents retinoicacid compounds from binding to Pin1 active site and induce Pin1degradation and to inhibit Pin1 function. Therefore, by detectingreduced Ser71 phosphorylation using phospho-specific Pin1 antibodiesthat we have generated can be a method to select patients for RAtreatments and to explain some patients may not respond to RA. Becauseaberrantly proliferating cells exhibit reduced Ser71 phosphorylation,these cells are more sensitive to RA treatments compared to normalcells.

The methods of the invention can also be used to detect geneticalterations in a Pin1 gene, thereby determining if a subject with thealtered gene is at risk for a disorder associated with the Pin1 geneand, consequently, a candidate for retinoic acid therapy. In preferredembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a Pin1-protein, or the mis-expression of the Pin1gene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a Pin1 gene; 2) an addition of one or morenucleotides to a Pin1 gene; 3) a substitution of one or more nucleotidesof a Pin1 gene, 4) a chromosomal rearrangement of a Pin1 gene; 5) analteration in the level of a messenger RNA transcript of a Pin1 gene, 6)aberrant modification of a Pin1 gene, such as of the methylation patternof the genomic DNA, 7) the presence of a non-wild type splicing patternof a messenger RNA transcript of a Pin1 gene, 8) a non-wild type levelof a Pin1-protein, 9) allelic loss of a Pin1 gene, and 10) inappropriatepost-translational modification of a Pin1-protein. As described herein,there are a large number of assay techniques known in the art which canbe used for detecting alterations in a Pin1 gene. A preferred biologicalsample is a tissue or serum sample isolated by conventional means from asubject, e.g., a cardiac tissue sample.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the Pin1-gene (seeAbravaya et al. (1995) Nucleic Acids Res 0.23:675-682). This method caninclude the steps of collecting a sample from a patient, isolatingnucleic acid (e.g., genomic, mRNA or both) from the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a Pin1 gene under conditions such that hybridization andamplification of the Pin1-gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. It is anticipated that PCR and/or LCR may be desirable to use asa preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a Pin1 gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in Pin1 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, geneticmutations in Pin1 can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin, M. T. et al. supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the Pin1 gene anddetect mutations by comparing the sequence of the sample Pin1 with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

Other methods for detecting mutations in the Pin1 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type Pin1 sequence with potentially mutant RNA orDNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to base pair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Nat Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control DNA or RNA can belabeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in Pin1 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a Pin1 sequence,e.g., a wild-type Pin1 sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in Pin1 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci. USA: 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control Pin1nucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. SciUSA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner et al. (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a Pin1 gene.

Furthermore, any cell type or tissue in which Pin1 is expressed may beutilized in the prognostic assays described herein.

As with the diagnostic assay described above, prognostic assays of Pin1activity can be included as part of a panel of target genes.

Additional methods of detecting Pin1 activity and diagnosing Pin1related disorders are disclosed in U.S. Patent Application PublicationNos.: 2009/0258352, 2008/0214470, 2006/0074222, 2005/0239095,US2002/0025521, U.S. Pat. No. 6,495,376, and PCT Application PublicationNo. WO02/065091, each of which is hereby incorporated by reference inits entirety.

The present invention also features methods and compositions todiagnose, treat and monitor the progression of a disorder describedherein (e.g., a cellular proliferation disorder) by detection andmeasurement of, for example, Pin1 substrates (or any fragments orderivatives thereof) containing a phosphorylated Ser/Thr-Pro motif in acis or trans conformation, as described in U.S. Patent ProvisionalApplication No. 61/255,431, which is hereby incorporated by reference inits entirety. The methods can include measurement of absolute levels ofthe Pin1 substrate (examples of which are listed in Tables 2, 3 and 4)in a cis or trans conformation as compared to a normal reference, usingconformation specific antibodies. For example, a serum level or level ina biopsy of a Pin1 substrate in the cis or trans conformation that isless than 5 ng/ml, 4 ng/ml, 3 ng/ml, 2 ng/ml, or less than 1 ng/ml serumor a biopsy is considered to be predictive of a good outcome in apatient diagnosed with a disorder (e.g., a disorder associated with aderegulation of Pin1 activity). A serum level of the substrate in thecis or trans conformation that is greater than 5 ng/ml, 10 ng/ml, 20ng/ml, 30 ng/ml, 40 ng/ml, or 50 ng/ml is considered diagnostic of apoor outcome in a subject already diagnosed with a disorder, e.g.,associated with a deregulation of Pin1 activity.

For diagnoses based on relative levels of substrate in a particularconformation (e.g., a Pin1 substrate in the cis or trans conformation),a subject with a disorder (e.g., a disorder associated with aderegulation of PPIase activity) will show an alteration (e.g., anincrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) in theamount of the substrate in, for example, the cis conformation. A normalreference sample can be, for example, a prior sample taken from the samesubject prior to the development of the disorder or of symptomssuggestive of the disorder, a sample from a subject not having thedisorder, a sample from a subject not having symptoms of the disorder,or a sample of a purified reference polypeptide in a given conformationat a known normal concentration (i.e., not indicative of the disorder).

Standard methods may be used to measure levels of the substrate in anybodily fluid, including, but not limited to, urine, blood, serum,plasma, saliva, amniotic fluid, or cerebrospinal fluid. Such methodsinclude immunoassay, ELISA, Western blotting, and quantitative enzymeimmunoassay techniques.

For diagnostic purposes, the conformation-specific antibodies may belabeled. Labeling of the antibody is intended to encompass directlabeling of the antibody by coupling (e.g., physically linking) adetectable substance to the antibody, as well as indirect labeling theantibody by reacting the antibody with another reagent that is directlylabeled. For example, the antibody can be labeled with a radioactive orfluorescent marker whose presence and location in a subject can bedetected by standard imaging techniques.

The diagnostic methods described herein can be used individually or incombination with any other diagnostic method described herein for a moreaccurate diagnosis of the presence or severity of a disorder (e.g., acellular proliferation disorder or a neurological disorder). Examples ofadditional methods for diagnosing such disorders include, e.g.,examining a subject's health history, immunohistochemical staining oftissues, computed tomography (CT) scans, or culture growths.

Monitoring the Effects of Retinoic Acid Treatment, and DiseaseProgression

In one embodiment, the present invention features a method formonitoring the effectiveness of treatment of a subject with a retinoicacid compound comprising the steps of (i) obtaining a pre-administrationsample from a subject prior to administration of the compound; (ii)detecting the level of expression or activity of a Pin1 protein, Pin1phosphorylation on Ser71, mRNA, or genomic DNA in the pre-administrationsample; (iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of the Pin1protein, mRNA, or genomic DNA in the post-administration samples; (v)comparing the level of expression or activity of the Pin1 protein, mRNA,or genomic DNA in the pre-administration sample with the Pin1 protein,mRNA, or genomic DNA in the post administration sample or samples; and(vi) altering the administration of the retinoic acid compound to thesubject accordingly. According to such an embodiment, Pin1 expression,phosphorylation or activity may be used as an indicator of theeffectiveness of the retinoic acid compound, even in the absence of anobservable phenotypic response.

In another embodiment, the diagnostic methods described herein can alsobe used to measure the levels of, for example, polypeptides (e.g., Pin1substrates listed in Tables 2, 3 and 4) with pSer/Thr-Pro motifs in thecis or trans conformation using conformation specific antibodies, Themethods can include repeated measurements, using conformation specificantibodies, for diagnosing the disorder and monitoring the treatment ormanagement of the disorder. In order to monitor the progression of thedisorder in a subject, subject samples can be obtained at several timepoints and conformation specific antibodies can be used to monitor thelevels of cis and trans isomers of Pin1 substrates (listed in Tables 2,3 and 4). For example, the diagnostic methods can be used to monitorsubjects during chemotherapy (e.g., therapy with a retinoic acidcompound or other agent described herein). In this example, serumsamples from a subject can be obtained before treatment with achemotherapeutic agent, again during treatment with a chemotherapeuticagent, and again after treatment with a chemotherapeutic agent. In thisexample, the level of Pin1 substrate with a pSer/Thr-Pro motif in thecis conformation in a subject is closely monitored using theconformation-specific antibodies of the invention and, if the level ofPin1 substrate with a pSer/Thr-Pro motif in the cis conformation beginsto increase during therapy, the therapeutic regimen for treatment of thedisorder can be modified as determined by the clinician (e.g., thedosage of the therapy may be changed or a different therapeutic may beadministered). The monitoring methods of the invention may also be used,for example, in assessing the efficacy of a particular drug or therapyin a subject, determining dosages, or in assessing progression, status,or stage of the infection.

TABLE 2 General Pin1 Substrates Substrate (GenBank FunctionalConsequence of PPIase Accession Number) Targeting Site(s) Activity ofPin1 Upon Substrate G2/M and Mitotic Regulation NIMA (P11837) —Regulation of mitotic function RAB4 (NP_004569) — — CDC25 (AAA58417)pThr48/67-Pro Dephosphorylation and regulation of activity WEE1(NP_003381) pT186-P Inhibition of WEE1 activity PLK1 (P53350) — — MYT1(NP_004194) — — CDC27 (AAH11656) — — CENP-F (P49454) — — Incenp(NP_064623) — — RPB1 (CAA65619) pSer5-Pro Regulation of CTDdephosphorylation NHERF-1 pSer279/301-P Dephosphorylation (AAA80218)KRMP1 (NP_057279) pT-1604-P Regulation of mitotic function CK2(NP_808227) Multiple pSer/Thr-Pro Inhibition of kinase activity sitesTopoIIα (NP_001058) — Inhibition or induction of phosphorylation DAB2(NP_001334) — Dephosphorylation p54nrb (CAA72157) Multiple pSer/Thr-Pro— sites Sil (CAC14001) Multiple pSer/Thr-Pro Regulation of functionsites EMI1 (NP_036309) pS10-P Stabilization G1/S Regulation Cyclin D1pT286-P Stabilization and nuclear localization (NP_444284) Ki67 pT234-P— c-Myc (CAA46984) pT58-P Dephosphorylation and destabilization Cyclin E(P24864) pS384-P Destabilization Growth and Oncogenic Signaling c-Jun(AAH06175) pS63/73-P Transactivation B-catenin (P35222) pS246-PStabilization, protein interaction, and transactivation Cf-2 (NP_034298)— Destabilization NF-κB (AAH33210) pT254-P Stabilization, proteininteraction, and transactivation RAF1 (AAA60247) Multiple pSer/Thr-ProDephosphorylation and prolonging sites activation c-Fos (CAA24756)Multiple pSer/Thr-Pro Transactivation sites RARα pS77-P Stabilizationand transactivation (NP_001019980) AIB1/SRC-3 — Transactivation anddestabilization HBx (NP_110380) pS41-P Stabilization and potentiationSTAT3 (NP_998827) pS727-P Transactivation DNA Damage, Oxidative StressResponse, and Apoptosis p53 (BAC16799) Multiple pSer/Thr-ProStabilization and transactivation sites Bcl-2 (NP_000648) pS70/87-P —p73 (CAA72221) Multiple pSer/Thr-Pro Stabilization and transactivationsites BimEL (AAC39593) pS65-P Stabilization p66^(shc) (AAH14158) —Mitochondrial import CHE1 (P06276) — Destabilization Neuronal Survivaland Degeneration Tau (NP_058519) pT231-P Dephosphorylation and proteinpT212-P interaction APP (P05067) pT668-P Promotes non-amyloidogenic APPprocessing and reduces Aβ production APP fragment pT668-P Increases Aβproduction from C99 APP fragment Synphilin-1 pS211/215-P Proteininteraction (AAD30362) Gephyrin (CAC81240) pS188/194/200-P Proteininteraction MCL1 (CAI15504) pT163-P Stabilization Immune Response andAsthma NFAT (NP_666017) — AUF1 (NP_112738) — Protein interaction IRF3(AAH71721) pS339-P Destabilization BTK (CAI42359) pS21/115-PDestabilization Others SIN2-RPD3 — Reduces histone deacetylases hSpt5(NP_001124297) —

TABLE 3A Pin1 targets where Pin1 prevents protein from degradationPATHWAYS/ PROTEIN PIN1 BINDING KINASE/ DISEASES/ NAME PIN1 FUNCTIONMOTIF PHOSPHATASE MECHANISM 1 p53 1. Stabilization; Increase Ser33,Ser315, Chk2 Genotoxic stress, DNA p53 promoter binding Thr181; Pro82damage activity Trophoblast invasiveness 2. Pin1 information of HMWC andstabilizes p53 2 Cyclin D1 Stabilization; localization Thr286 BlockSocs-1 ub- Cell proliferation/cancers and transcription mediatedproteolysis 3 Tau 1. Dephosphorylated at Thr231 Tauopathy Thr231 2. Pin1knockdown or KO increased WT tau protein stability in vitro 4 β-CateninStability; localization and Ser246 Could stabilize β- Cellproliferation/cancers transactivation catenin by inhibiting GSK-3βdependent degradation 5 c-Jun Transcriptional activity Ser63/Ser73 INKBreast cancer; AML 6 p65/NF-kB Nuclear translocation; Thr254 Cytokines;Hepatocyte stability NF-kappaB activation 7 p73 Stabilizing,transcriptional Ser412, Thr442 c-Abl and p300 Genotoxic stress activityand Thr482 8 Synphilin-1 Facilitates Lewy Body Ser211 and casein kinaseII Parkinson disease formation; stabilizes Ser215 alpha-synuclein 9c-Fos Transcriptional activation C-terminal ERK 10 Sil No impact on Silspindle Unknown checkpoint 11 p54nrb Thr412, Thu-430 Cdk1 and Thr452 12Bruton Mediates Btk degradation Ser21 an Ser115 Tyrosine kinase tyrosinekinase (Btk) 13 AUF1 Regulates GM-CSF Ser83 mRNA; AUF1, AU rich element-binding protein 14 BIM_(EL) Stabilize BIM_(EL) and Ser65 JIP3, MKK7 andJNK Neuronal apoptosis induce apoptosis 15 Mcl-1 1. Pin1 inhibits Mcl-1Thr163; Thr92 JNK3 induces Mcl-1 Oligodendrocyte (Myeloid cellubiquitination degradation by counting apoptosis; Mcl-1 leukemia 2.Stabilizes Mcl-1 the protective binding of mediated sequence-1) Pin1chemoresistance; breast Erk phosphorylates cancer Thr92 and Thr163 16HBx; Pin1 overexpression Ser41 Pin1 binds HBx and HepatocarcinogenesisHepatitis B increased the protein enhance virus encoded stability of HBxhepatocarcinogenesis in protein X HBV-infected hepatocytes 17 OriginPrevents degradation of APC; Topo II Mitotisis; chromosome recognitionORC1 by inactivating segregation and for complex, mitotic APC complexreprogramming replicons subunit 1 (ORC1) 18 Bcl-2 1. Induce changes inthe Possibly mediated by bioactivity of Bcl-2; cdc2 2. Preventsdephosphorylation of Bcl-2 19 Erb2 Stabilize ErbB2 Ubiquitinated ErbB2pathway; Her2-positive breast erbB2 ubiquitin mediated cancerdegradation 20 PPARγ Prevents the Ser84 Ras mediated kinase Macrophagesmediated polyubiquitination of atherosclerosis PPARγ through ubiquitin-proteosome pathway 21 Cep55 Increased Cep55 stability Ser425, Ser428Cdk1, Plk1 Mitosis and cytokinesis 22 Spt23 Ess1 stabilizes Spt23 Ser654Unsaturated fatty-acid synthesis 23 p27 Pin1 protects p27 from Thr187Cdk2 Cell cycle; Cancer degradation through polyubiquitinationmechanism. 24 Akt Regulates Akt protein Thr92, Thr450 Oncogenesisstability 25 HTLV Tax Increased Tax protein Ser160 Pathogenesis of Humanprotein expression; inhibits Tax T-cell leukemia virus type proteindegradation 1 (HTLV-1) related diseases 26 Nanog Leads to Nanogstability Ser52, Ser65 Stem cells pluripotency; cell renewal 27 ViralStabilized phospho-HIV-1 Ser57 JNK HIV-1 cDNA integration Integraseintegrase and infection

TABLE 3B Pin1 targets where Pin1 enhances degradation of phosphorylatedproteins PATHWAYS/ PROTEIN PIN1 BINDING KINASE/ DISEASES/ NAME PIN1FUNCTION MOTIF PHOSPHATASE MECHANISM 1 c-Myc Enhances c-Myc Thr58Tumorgenesis degradation 2 Cyclin E Depletion of Pin1 Ser384 CyclinE-Cdk2 complex Cell cycle, genomic upregulates cellular levelinstability and of cyclin E tumorigenesis 3 SRC-3/AIB1 Enhances SRC-3N/A Steroid receptor degradation 4 RARα Induces its degradation Ser77Retinoic acid receptor; 5 IRF3 Promote its degradation Ser339 Hostantiviral responses via the ubiquitin- during virus infection proteasomepathway. 6 Che-1 Conformational changes Thr144 p53 transcription; DNAinduced by Pin1 are damage apoptotic requested for Che- pathway 1/HDM2interaction 7 Pim-1 protein Binding of Pin1 leads to a N/A PP2A Elevatedin lymphomas kinase decrease in the protein leukemias and prostate levelof Pim-1 cancer through c-Myc pathway 8 Promyelocytic Binds tophosphorylated C-terminal of Breast cancer; hydrogen leukemia C terminusof PML and PML (Ser403, peroxide-induced death; protein enhances PMLSer505, Ser518 cell proliferation (PML) degradation and Ser527) 9 FOXO Anovel negative FOXO Oxidative stress regulator, interconnecting FOXOphosphorylation and monoubiquitination in response to cellular stress toregulate p27 10 Silencing Pin1 destabilizes SMRT Ser1241, Her2/Neu/ErbB2Human cancer mediator for Thr1445, Ser1469 receptor; Cdk2 retinoic acidand thyroid hormone receptor (SMRT) 11 TRF-1 Pin1 inhibition resulted inThr149 Cdk Cancer; ageing decrease TRF1 degradation; 12 G protein-Promotes GRK2 Ser670 CDK2-cyclinA Cell cycle progression; coupleddegradation p53 response and the receptor induction of apoptosis kinase2 (GRK2) 13 SF-1 Pin1 promotes SF-1 Ser203 CDK7 Gonadotropin beta-ubiquitination and subunit gene transcription degradation 14Sulfotransferase Pin1 destabilizes Thr8, Thr11 ERK1 and PP2A Metabolismof 4A1 SULT4A1 endogenous and (SULT4A1) exogenous compounds 15 Smad2/Reduced Smad2/3 protein Thr179, Ser204, Smurf2 with Smads and TGF-βsignaling Smad3 levels Ser208, Ser213 enhanced Smad (Smad3 linkerubiquitination domain) 16 MEF2C Pin1 decreases MEF2C Ser98/Ser110 Muscleterminal stability differentiation

TABLE 3C Pin1 targets where Pin1 regulates targetphosphorylation/dephosphorylation/other modifications PATHWAYS/ PROTEINPIN1 BINDING KINASE/ DISEASES/ NAME PIN1 FUNCTION MOTIF PHOSPHATASEMECHANISM 1 Tau Enhance Thr231 Neuronal differentiation;dephosphorylation at stress induced; Thr231 Alzheimer disease 2 Raf-1Dephosphorylation by N.A. Ras/MAP Kinase PP2A 3 Type-1 Cyclingphosphorylation Thr72 CDK1-cyclin B Entry and exit in mitotic protein inmitotic phosphatase Inhibitor-2 (I- 2) 4 TGF-β1 Decay, accumulation andChronic asthma mRNA translation of TGF-β1 mRNA in Eosinophils 5K-homology parathyroid hormone Ser181 splicing (PTH) mRNA stabilityregulator protein (KSRP)

TABLE 4 Pin1-targets for promoting tumorigenesis Activation/ SubstratesFunction Binding Sites Effects Inactivation AIB1/SRC3 Transactivator —Activity + Akt Protein kinase pThr^(92/450)-Pro Stability + BaxApoptosis pThr¹⁶⁷-Pro Activity − Bcl-2 Antiapoptic protein — Stability −Btk Tyrosine kinase pSer^(21/115)-Pro Stability − β-cateninTranscription factor pSer²⁴⁶-Pro Localization, stability + C/EBPTranscription factor — Activity − Cyclin D1 Transcription factorpThr²⁸⁶-Pro Localization, stability + Daxx Apoptosis pSer¹⁷⁸-ProStability − FAK Tyrosine kinase pSer⁹¹⁰-Pro Activity + c-FosTranscription factor — Activity + FOXO4 Transcription factor —Localization, activity − GRK2 G protein receptor pSer⁶⁷⁰-Pro Stability −Hbx Transactivator pSer⁴¹-Pro Activity, stability + c-Jun Transcriptionfactor pSer^(63/73)-Pro Activity, stability + Mcl-1 ApoptosispThr^(92/163)-Pro Stability + c-Myb Transactivator pSer⁵²⁸-ProActivity + Neu Growth factor receptor — Stability + NF-κB Transcriptionfactor pThr²⁵⁴-Pro Localization, stability + Notch1 Growth factor —Activity + p70S6K Ribosomal S6 kinases — Activity + p53 Transcriptionfactor — Activity, stability −* Plk1 Mitotic kinase — Binding activity +PML Transcription factor pSer^(403/505/518/527)- Stability − Pro Raf-1Protein kinase — Activity + RARα Transcriptional regulator pSer⁷⁷-ProStability − V-Rel Transcription factor pThr²⁵⁴-Pro Localization,stability + Smad Transactivator — Stability − SMRT Transcriptionalco-repressor pSer^(1241/1469), Stability − Thr¹⁴⁴⁵-Pro Stat3Transcription factor pSer⁷²⁷-Pro Activity + Tax Viral oncoproteinpSer¹⁶⁰-Pro Activity, stability + Pin2/TRF1 Telomere regulationpThr¹⁴⁹-Pro Stability −

III. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) aproliferative disorder (e.g., a disorder associated with increased Pin1expression or activity) with a retinoic compound.

Certain embodiments of the invention feature formulation of a retinoicacid compound for, e.g., controlled or extended release. Many strategiescan be pursued to obtain controlled and/or extended release in which therate of release outweighs the rate of metabolism of the therapeuticcompound. For example, controlled release can be obtained by theappropriate selection of formulation parameters and ingredients (e.g.,appropriate controlled release compositions and coatings). Examplesinclude single or multiple unit tablet or capsule compositions, oilsolutions, suspensions, emulsions, microcapsules, microspheres,nanoparticles, patches, and liposomes. The release mechanism can becontrolled such that the retinoic acid compound is released at periodintervals, the release could be simultaneous, or a delayed release ofone of the agents of the combination can be affected, when the earlyrelease of one particular agent is preferred over the other.

Certain embodiments of the invention feature a deuterated retinoic acidcompound that is made by replacing some or all hydrogen with deuteriumusing state of the art techniques (e.g., as described herein and atwww.concertpharma.com).

Prophylactic Methods

In one aspect, the invention provides a method for preventing aproliferative disorder in a subject by administering to the subject aretinoic acid compound. Subjects at risk for a disease which is causedor contributed to by aberrant Pin1 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a retinoic acidcompound can occur prior to the manifestation of symptoms characteristicof the Pin1 aberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression.

Combination Therapies

Anti-proliferative and other anti-cancer compounds (e.g.,anti-angiogenic compounds) are useful for treating proliferativedisorders in combination with the retinoic acid compounds of theinvention.

Anti-proliferative agents that can be used in combination with aretinoic acid compound include, without limitation, microtubuleinhibitors, topoisomerase inhibitors, platins, alkylating agents, andanti-metabolites. Particular anty-proliferative agents that are usefulin the methods and compositions of the invention include, withoutlimitation, paclitaxel, gemcitabine, doxorubicin, vinblastine,etoposide, 5-fluorouracil, carboplatin, altretamine, aminoglutethimide,amsacrine, anastrozole, azacitidine, bleomycin, busulfan, carmustine,chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine,cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin,daunorubicin, docetaxel, estramustine phosphate, floxuridine,fludarabine, gentuzumab, hexamethylmelamine, hydroxyurea, ifosfamide,imatinib, interferon, irinotecan, lomustine, mechlorethamine, melphalen,6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone,pentostatin, procarbazine, rituximab, streptozocin, tamoxifen,temozolomide, teniposide, 6-thioguanine, topotecan, trastuzumab,vincristine, vindesine, and vinorelbine. The ability of a compound toinhibit the growth of a neoplasm can be assessed using known animalmodels.

Compounds which are known to interact with other proteins implicated inPin1 signaling pathways can also be useful in combination with aretinoic acid compound (see, e.g., the targets and compounds in Table5).

TABLE 5 Target Target Class Representative Antagonist AKT Kinase MK-2206Cyclin D1 Cyclin ON 013105 HER2/Neu (ErbB-2) Kinase Herceptin NF-□FTranscription Factor RTA 402 Plk Kinase BI 2536 Raf-1 Kinase SorafenibStat3 Transcription Factor ISIS-STAT3Rx □SIS-STAT Adhesion NucleicAcid-Based Rx in Enzon Program at Santaris

Such compounds can act synergistically with a retinoic acid compound.Additionally, co-administration with a retinoic acid compound may resultin the efficacy of the anti-proliferative compound at lower (and thussafer) doses (e.g., at least 5% less (e.g., at least 10%, 20%, 50%, 80%,90%, or even 95%) less than when the anti-proliferative compound isadministered alone.

Therapy according to the invention may be performed alone or inconjunction with another therapy and may be provided at home, thedoctor's office, a clinic, a hospital's outpatient department, or ahospital. Treatment optionally begins at a hospital so that the doctorcan observe the therapy's effects closely and make any adjustments thatare needed, or it may begin on an outpatient basis. The duration of thetherapy depends on the type of disease or disorder being treated, theage and condition of the patient, the stage and type of the patient'sdisease, and how the patient responds to the treatment. Additionally, aperson having a greater risk of developing an proliferative disease mayreceive treatment to inhibit or delay the onset of symptoms.

Routes of administration for the various embodiments include, but arenot limited to, topical, transdermal, nasal, and systemic administration(such as, intravenous, intramuscular, subcutaneous, inhalation, rectal,buccal, vaginal, intraperitoneal, intraarticular, ophthalmic, otic, ororal administration). As used herein, “systemic administration” refersto all nondermal routes of administration, and specifically excludestopical and transdermal routes of administration.

In combination therapy (e.g., a retinoic acid compound with a secondanti-proliferative agent), the dosage and frequency of administration ofeach component of the combination can be controlled independently. Forexample, one compound may be administered three times per day, while thesecond compound may be administered once per day. Alternatively, onecompound may be administered earlier and the second compound may beadministered later. Combination therapy may be given in on-and-offcycles that include rest periods so that the patient's body has a chanceto recover from any as yet unforeseen side effects. The compounds mayalso be formulated together such that one administration delivers bothcompounds.

Each compound of the combination may be formulated in a variety of waysthat are known in the art. For example, the first and second agents maybe formulated together or separately. Desirably, the first and secondagents are formulated together for the simultaneous or near simultaneousadministration of the agents. Such co-formulated compositions caninclude the two drugs together in the same pill, ointment, cream, foam,capsule, liquid, etc. It is to be understood that, when referring to theformulation of combinations of the invention, the formulation technologyemployed is also useful for the formulation of the individual agents ofthe combination, as well as other combinations of the invention. Byusing different formulation strategies for different agents, thepharmacokinetic profiles for each agent can be suitably matched.

The individually or separately formulated agents can be packagedtogether as a kit. Non-limiting examples include kits that contain,e.g., two pills, a pill and a powder, a suppository and a liquid in avial, two topical creams, ointments, foams etc. The kit can includeoptional components that aid in the administration of the unit dose topatients, such as vials for reconstituting powder forms, syringes forinjection, customized IV delivery systems, inhalers, etc. Additionally,the unit dose kit can contain instructions for preparation andadministration of the compositions. The kit may be manufactured as asingle use unit dose for one patient, multiple uses for a particularpatient (at a constant dose or in which the individual compounds mayvary in potency as therapy progresses); or the kit may contain multipledoses suitable for administration to multiple patients (“bulkpackaging”). The kit components may be assembled in cartons, blisterpacks, bottles, tubes, and the like.

IV. Experimental Results

We have identified a series of nanomolar inhibitors that binds only tothe catalytically active PPIase domain of Pin1. Upon electroporation ormicroinjection, these Pin1 peptide inhibitors block HeLa division, andthis block is rescued by co-injected Pin1, indicating that the compoundsare highly specific and potent. Using the above Pin1 peptide inhibitors,we have developed a high-throughput screen for identifying Pin1inhibitors using a single-step fluorescence polarization-baseddisplacement assay (FP-HTS). The FP-HTS detects molecules that competefor the substrate binding to the catalytic active site, measures ligandbinding under equilibrium conditions, and does not suffer from productinhibition. The HF488 fluorescent probes for the FP assay contain onlyfour residue core structure of Bth-L-phos.Thr-Pip-NaI (pTide), with a Kdof 258 nM for Pin1, was synthesized by Anaspec. We performed FP-HTS in a384-well plate format with full length Pin1 and produced robust FP,resulting in a 6-7 fold increase in polarization degree values, using aSynergy II plate reader. This novel FP-HTS showed robust andreproducible performance. The assay can tolerate up to 10% DMSO. The Z′is around 0.70 and is consistent for day-to-day performance. Thecoefficient of variation is in the range of 4-5%. More importantly, wehave shown that both pTide, the unlabeled Pin1 peptide as the positivecontrol in this project, and juglone, the Pin1 small molecule inhibitor,displaced HF488 probe from Pin1. Although it is difficult to determinethe Kd for the covalent and irreversible inhibitor juglone, the Kd forpTide was ˜250 nM, similar to that derived from PPIase-based assays.

We used the FP-HTS with a 5 nM probe and 200 nM Pin1 to conduct a pilotscreen on a selected set of chemical libraries. We obtained theresulting potential positive hits and grouped them into 3 classesaccording to the Z-score, which is folds of standard deviation below themean. The top strong chemical is the clinically used drug13-cis-retinoic acid (cis-RA) (FIG. 2A). cis-RA is particularlyattractive for the following reasons. 1) cis-RA is not listed inpromiscuous inhibitor databases 2) all-trans retinoic acid (trans-RA),the pairing compound of cis-RA, is currently used as oral prescriptionfor patients with acute promyelocytic leukemia (AML); 3) both cis-RA andtrans-RAs are used as a medication in the phase II/III clinical trialfor breast cancer, and more because of its ability to kill rapidlydividing cells; and 4) although it has been reported that RAs target onretinoic acid receptor (RAR), the exact mechanism of the anticanceraction is unknown. As a result, we examined whether the anti-cancereffect of RAs depends on RAR in the breast cancer cells. RARα knockdowncan only partially rescue-cis-RA-mediated cell death, indicating thatRAs may have unidentified “off-target effect.” To confirm that RAsindeed target Pin1, we examined cis-RA and trans-RA in the FP assay,and, surprisingly, found that trans-RA displayed even more prominentPin1 inhibition than cis-RA dose and that cis-RA would eventually catchup with trans-RA in the long-term incubation with Pin1, likely due toresonance-mediated cis-trans conversion (FIG. 3). In the PPIase assay,Pin1 activity was blocked by either cis- or trans-RA in a dose-dependentmanner (FIG. 4). These data confirm that the interaction between RAs andPin1 is specific and not due to aggregation. Furthermore, both trans andcis RA blocked the association between Pin1 and DAPK1 in adose-dependent manner, with trans being more potent (FIG. 5). Theseresults indicate that RAs binds to Pin1 C-terminal catalytic domainbecause DAPK1 is known to bind this domain (Lee et al., 2011 Mol Cell inpress). To determine which amino acid residue in the Pin1 catalyticdomain that are important for retinoic acid binding, point mutations ofPin1 including K63A, S67E, R68/69A, H59A or S71E completely orsignificantly abolished trans-RA binding to Pin1 (FIGS. 6A and B).Together, these data indicate that RAs inhibit Pin1 by occupying itscatalytic PPIase pocket in the C-terminus and that phosphorylation ofSer67 or Ser71 inhibits RA binding to Pin1.

To further discern the causal relationship between theanti-proliferative effect of RAs on Pin1, cell viability of three breastcancer cell lines was tested with different dosages of cis- or trans-RA,of which SKBR3 and T47D exhibited preferential sensitivity to RAs withan IC50 in the nano-molar range, while the normal cell line MCF10Aremained unaffected (FIG. 7A). This discrepancy between cell lines wascorrelated with the RAs' ability to suppress Pin1 expression. Pin1 levelwas decreased by treatment of RAs in drugs-responsive SKBR3 and T47D,but not in drugs-irresponsive normal cells, MCF10A (FIG. 7B), in whichPin1 target protein, cyclin D1, was served as biomarkers of in vivo Pin1activity. Moreover, RAs did not alter Pin1 mRNA level, but did reducePin1 protein stability, suggesting that RAs interact with Pin1, resultin Pin1 degradation, and subsequently leads to anti-proliferation ofbreast cancer cells (FIGS. 8A-C). To further confirm this premise,wild-type mouse embryonic fibroblast (WT MEF) and Pin1 knockout MEF(Pin1 KO MEF) were used to test trans-RA-mediated cell viability (FIGS.9A-C). As expected, Pin1 KO MEF was more resistant to trans-RA than WTMEF due to lack of drug target. In addition, Pin1 KO MEF stablyexpressing WT Pin1, but not W34/K63A Pin1 mutant, enabled cells tore-sensitize trans-RA. These results indicate that RAs-mediated celldeath is at least in part dependent on Pin1.

As trans-RA is selected as the current lead compound, it is important toidentify the essential moiety of RAs on Pin1 interaction for furtherlead optimization. Many commercially available retinoids were tested forPin1 inhibition. Only those with end carboxylic group maintained Pin1inhibition in a cell culture model, which are cis-RA, trans-RA,AC-55649, and acitretin (FIGS. 10A, 10B, 11A, and 11B). Other RAcompounds that are metabolized to contain an end carboxylic group arealso likely to be useful in treating proliferative disorders (FIG. 10D).In addition, compounds that modulate the retinoic acid receptor (FIGS.10D and 11A and compounds listed in Table 1) are also likely to beuseful in treating proliferative disorders.

In addition, we have developed Cell-based assays to screen and validatePin1 inhibitor hits. We have shown 1) that Pin1 is highly expressed inHER2-positive human breast cancer tissues; 2) that Pin1 inhibitionalmost completely suppresses HER2 overexpression on cell surface inhuman HER2+ breast cancer cell lines such as AU565 and SKBR3 cells; 3)that Pin1 inhibition greatly increases the sensitivity of HER2+ breastcancer cells to the mTOR inhibitor, but not to the HER2 inhibitor,suggesting that Pin1 might act on Her2 to regulate cell growth; 4) thatPin1 acts on Neu and multiple substrates in Neu-mediated oncogenicpathway; and 5) that Pin1 knockout in mice inhibits breast cancerdevelopment induced by activated Her2. Therefore, Pin1 is essential formaintaining HER2 overexpression and growth of human HER2+ breast cancercells. Given that HER2 expression on cell surface and cell growth arereadily assayed on 384-well format, we can test the ability of the hitsto repress HER2 overexpression and cell growth of HER2_AU565 and SKBR3cells, which will be treated with Pin1 prodrugs or hits, and thenimmunostained with Alexa 488-anti-HER2 monoclonal antibody (BioLegend),followed by automated microscopy.

We have further shown that combinations of therapeutic compoundsincluding retinoic acid compounds are useful for treating cancer, e.g.,cancer characterized by elevated Pin1 activity. FIG. 12 shows resultsobtained from the treatment of breast cancer cells overexpressing Pin1with ATRA or Doxorubicin or their combination, followed by countingcancer cell numbers. The results show that ATRA and Doxorubicincombination dramatically increases anticancer potency and reduce thedose of each drug to inhibit cancer cell growth. Therefore, ATRA candrastically reduce dose and toxicity of Doxorubicin and otherchemotherapeutic drugs.

We have also shown that Pin1 inhibition, using siRNA, dramaticallyreduces Neu/Erb2 overexpression and cell proliferation of human breastcancer cells that have Neu/Erb2 gene amplification (FIGS. 13A and 13B).This provides us with a method to identify a Pin1 modulatory compound byapplying a test compound to human-derived cancer cells that haveNeu/Erb2 gene amplification, and determining the effect of the testcompound on Neu/Erb2 overexpression and cell proliferation.

Other Embodiments

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth, and follows in the scope ofthe claims.

Other embodiments are within the claims.

What is claimed is:
 1. A method of treating a proliferative disease in asubject, said method comprising administering a retinoic acid compoundto said subject in an amount sufficient to treat said subject, whereinsaid subject is determined to have elevated levels of a Pin1 markerprior to said administration.
 2. A method of treating a proliferativedisease in a subject, said method comprising determining Pin1 markerlevels in a sample from said subject and administering a retinoic acidcompound to said subject if sample is determined to have elevated Pin1marker levels.
 3. The method of claim 1, further comprising theadministration of a second anti-proliferative compound.
 4. The method ofclaim 1, wherein said Pin1 marker is reduced Ser71 phosphorylation ofPin1.
 5. The method of claim 1, further comprising determining Pin1marker levels in said sample after said administration of a retinoicacid compound.
 6. The method of claim 1, wherein said retinoic acidcompound is selected from the group consisting of 13-cis-retinoic acidand all-trans-retinoic acid.
 7. The method of claim 1, wherein saidretinoic acid compound is selected from the group consisting of retinol,retinyl acetate, retinal, and AC-55640.
 8. The method of claim 1,wherein said retinoic acid compound is selected from compoundsstructurally similar to retinoic acid, preferably those listed in FIGS.2A, 2B, 10A, 10D, and Table
 1. 9. The method of claim 1, wherein saidretinoic acid compound is deuterated.
 10. The method of claim 1, whereinsaid sample is selected from the group consisting of tumor samples,blood, urine, biopsies, lymph, saliva, phlegm, and pus.
 11. The methodof claim 1, wherein said elevated Pin1 marker level is due to aninherited trait or a somatic mutation.
 12. The method of claim 3 whereinsaid second anti-proliferative compound is selected from the groupconsisting of MK-2206, ON 013105, RTA 402, BI 2536, Sorafenib, andISIS-STAT3Rx.
 13. The method of claim 3, wherein said secondanti-proliferative compound is selected from the group consisting ofmicrotubule inhibitors, topoisomerase inhibitors, platins, alkylatingagents, and anti-metabolites.
 14. The method of claim 3, wherein saidsecond anti-proliferative compound is selected from the group consistingof paclitaxel, gemcitabine, doxorubicin, vinblastine, etoposide,5-fluorouracil, carboplatin, altretamine, aminoglutethimide, amsacrine,anastrozole, azacitidine, bleomycin, busulfan, carmustine, chlorambucil,2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide,cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel,estramustine phosphate, floxuridine, fludarabine, gentuzumab,hexamethylmelamine, hydroxyurea, ifosfamide, imatinib, interferon,irinotecan, lomustine, mechlorethamine, melphalen, 6-mercaptopurine,methotrexate, mitomycin, mitotane, mitoxantrone, pentostatin,procarbazine, rituximab, streptozocin, tamoxifen, temozolomide,teniposide, 6-thioguanine, topotecan, trastuzumab, vincristine,vindesine, and vinorelbine.
 15. The method of claim 3, wherein saidanti-proliferative compound is administered at a low dosage or atdifferent time.
 16. The method of claim 3, wherein saidanti-proliferative compound is formulated in such as a liposomalformulation or a controlled release formulation.
 17. The method of claim3, wherein said retinoic acid compound and said anti-proliferativecompound are formulated together.
 18. The method of claim 1, whereinsaid proliferative disorder is selected from the group consisting ofleukemias, polycythemia vera, lymphomas, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors.
 19. The methodof claim 18, wherein said proliferative disorder is selected from thegroup consisting of acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, acute myeloblastic leukemia, acute promyelocyticleukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemi), Hodgkin's disease, non-Hodgkin's disease,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma.
 20. A methodfor identifying a Pin1 ligand comprising of: (i) incubating Pin1 proteinwith a fluorescently labeled probe, forming a Pin1-probe complex; (ii)adding test compound to the incubation; and (iii) determining whetherany substantial portion of the probe is displaced from the Pin1-probecomplex by the test compound, such displacement indicating that the testcompound is a Pin1 ligand.
 21. A method for identifying a Pin1modulating compound comprising of: (i) incubating human-derived cancercells that have Neu/Erb2 gene amplification; (ii) applying a testcompound to the cell; and (iii) determining whether Neu/Erb2overexpression is reduced, wherein reduction of Neu/Erb2 overexpressionindicates that the test compound is a Pin1 modulating compound.